Background: The prognosis of allogeneic hematopoietic stem cell transplantation (HSCT) for non-remission hematological malignant diseases is usually unfavorable. The most uncontrollable factor is residual disease or relapse. To overcome this problem, intensified conditioning regimens- sequential and/or additional chemotherapy to the standard regimen- could be effective. However, increasing the intensity of conditioning might also lead to more complications. Materials and Methods: We retrospectively analyzed 81 patients with non-remission disease who received allogeneic HSCT in our institution between 2007 and 2011. Results: 55.6% in 36 myeloablative conditioning patients and 46.7% in 45 reduced-intensity conditioning patients received intensified conditioning. The 5-year probability of overall survival was 35.0% and 17.1% in the standard and intensified group, respectively (p=0.027). Relapse mortality was 30% in the standard regimen group and 36.6% in the intensified regimen group (p=0.54). Transplant-related mortality (TRM) at 30 and 100 days was 5%, 17.1% (p=0.086) and 27.5%, 34.2% (p=0.52) in the standard and intensified group, respectively. There was no difference in TRM between the 2 groups at 30 days and 100 days.Conclusion: The results of the study confirm the safety of the intensified conditioning regimen. Meanwhile, it could be considered as one of the few methods available to reduce the tumor burden before HSCT for refractory malignant diseases. However, supportive therapies for allogeneic HSCT have recently been developed and to some extent, complications might be prevented with more effective drugs, anti-biotic, fungal, viral and graft-versus-host diseases (GVHD) drugs. The problem of residual disease or relapse remains unresolved. There are few therapeutic options available to address this significant problem. Basically, pre-HSCT conditioning regimens are fixed due to the limiting dose of each of the chemotherapeutic drugs and total body radiation needed to avoid organ failure, except bone marrow failure 7, 8. Intensified conditioning regimens for allo- HSCT have been reported in the past. Several studies have indicated that intensified conditioning regimens did not achieve higher overall survival (OS) due to an increase in transplant-related mortality (TRM) 9-13. A few have reported that additional chemotherapy drugs might improve the outcome 14-21. Decreasing the tumor burden before HSCT, using sequential and/or additional chemotherapy to the standard conditioning regimen might be effective for advanced disease; indeed, this has been considered at our institute. On the other hand, increasing the intensity of conditioning also might lead to more patient complications such as organ failure and infection. In this report, we analyzed the safety and effects of intensified conditioning regimens. The prognosis of allogeneic hematopoietic stem cell transplantation (HSCT) for non-remission hematological malignant disease is usually unfavorable due to the uncontrollable nature of the disease and the development of several complications PatientsPatient characteristics for the analysis are summarized in 22 was 0 in 26, 1 in 5, 2 in 4, 3 in 4 and 4 in 1 patient of the standard regimen group and 0 in 21, 1 in 9, 2 in 2 and 3 in 7. Infectious complications were determined in 16 patients in the standard group and 23 patients in the intensified group. Liver dysfunction evaluated by T-bil was mild (>ULN to 1.5×ULN) in 4 and moderate (>1.5×ULN) in 1 patient in the standard group, mild in 4 and moderate in 1 patient in the intensified group. It was also done by ALT: mild (>ULN to 2.5×ULN) in 0, moderate (>2.5× ULN ) in 2 patients in the standard group; mild in 2, moderate in 1 patient in the intensified group. Renal dysfunction was tested by Creatinine level: mild (1.2-2mg/dl) in 1, moderate (>2mg/dl) in 0 patient in the standard group and mild in 3 patients and moderate in 0 patient in the intensified group. Definition of comorbidities was referred to HCI-CI22. Regarding the donor types, HLA-matched siblings were 6, HLA-matched unrelated 29, HLA-mismatched siblings 5 , HLA-mismatched unrelated 11 and CB 30. Stem cell sources were: BM for 40 patients, CB for 30 patients and PB for 11 patients. There were 36 and 45 patients who received myeloablative conditioning (MAC) and reduced-intensity conditioning (RIC), respectively. We chose MAC regimen for patients who were under 59 years and RIC regimen for those who were over 60 years. Patients who underwent allo-SCT more than two times were excluded from the study. This study was approved by Ethics Committee of Hamanomachi Hospital.More than 30% blast cells in bone marrow was confirmed in 2 out of 8 of patients in the standard group, 15 out of 25 patients in the intensified group of AML, 1 out of 2 patients in the standard group and 1 out of 2 patients in the intensified group of ALL. Those classified as more severe than stable diseases (SD) status were: 9 out of 19 in the standard group, 2 out of 5 in the intensified group of ML, 2 out of 4 in the standard group, and all 6 in the intensified group of ATLL. Performance states (PS) were: 0 in 28 patients, 1 in 42 patients, 2 in 7 patients, 3 in 3 patients and 4 in 1 patient. Hematopoietic cell transplantation-comorbidity index (HCT-CI) Standard conditioning regimens and GVHD prophylaxis2×5 days + Melphalan (Mel) 40 mg/m2 ×2 days +TBI 2–4Gy were used as standard conditioning regimens. MAC was used for 36 patients and RIC for 45 patients. Intensified conditioning was given to 55.6% (20/36) in the MAC group and 46.7% (21/45) in the RIC group. Prophylaxis of GVHD was done by calcineurin inhibitor (tacrolimus or cyclosporin) with short-term methotrexate  or with mycophenolate mofetil  23, 24.For MAC, total body irradiation (TBI) 4 Gy×3 days + cyclophosphamide (CY) 60 mg/kg ×2 days or Busulfan (Bu) 4 mg/kg/day×4 days + CY 60 mg/kg × 2 days　were used as standard conditioning regimens. For RIC, Fludarabine (Flu) 30 mg/m2×6 days + Bu 3.2 mg/kg/day ×2–4 days + TBI 2–4Gy or Flu 25 mg/mIntensified conditioning regimens: sequential and/or additional chemotherapy to the standard conditioning regimens25, 26.We defined intensified conditioning regimens as the regimens which were strengthened by adding sequential and/or additional chemotherapy to the standard conditioning regimens. The sequential conditioning regimen was defined as starting the standard conditioning regimens at a nadir before adequate hematopoietic recovery so that the numbers of white blood cells were less than 1000/μl following the most recent chemotherapy. The additional conditioning regimen was defined as added some chemotherapeutic drugs, Ara-C, VP-16, anthracyclines and monoclonal antibodies within 2 days of the standard conditioning regimen for MAC or RIC. The complete sequential regimens were given to 29 patients, the additional regimens were given to 20 patients and both were given to 8 patients. The details of the added chemotherapies are shown in Statistical analysis27.Differences in Patients’ characteristics were analyzed by the Student’s t-test. Overall survival and progression -free survival were calculated from the date of stem cell transplantation using the Kaplan-Meier product-limit method, and the difference between the groups was assessed using the log-rank test. Cumulative incidence of transplant-related mortality was evaluated by the Gray test. All statistical analyses were performed using EZR version 1.30  EngraftmentNeutrophil engraftment was achieved in 85.4% patients at a median of 18.1 days following transplantation (range: 9 – 43 days) in the intensified group and in 90.0% patients at a median of 19.0 days following transplantation (range: 14 – 42 days) in the standard group. In total, 3 patients experienced primary graft failure. Of 2 patients in the intensified group, 1 had HLA antibodies and the reason for the other one was unknown. The graft of 1 patient in the standard group was also failed for unknown reason. Platelet engraftment of ≧20,000/μl was achieved in 58.5% patients in a median of 40 days (range: 12 – 270 days) in the intensified group and in 70% patients in a median of 31.6 days (range: 13 – 107 days) in the standard group.Acute GVHDp=0.446, 0.568) 28. No statistically significant differences were observed. The cumulative incidences of acute GVHD in grade 1–4 and 3–4 were 65.9% and 17.0% in the intensified group and 57.5% and 12.5% in the standard group, respectively  (p=0.099) (p=0.036) . The Kaplan-Meier estimate of 5-year OS was 17.1% (7.5-30.0%) for the intensified group and 35.0% (20.8-49.6%) for the standard group (p=0.027) . Resultsp=0.099) . Progresp=0.036) . The resTransplant-related toxicity (TRT)29. Zero organ was 39%, one was 29.3%, two was 24.4%, three was 7.3% and more Transplant-related toxicity, non-hematopoietic side effects of main organ dysfunction , more than grade 3, was analyzed until day 100. The common terminology criteria (CTC) for adverse events, version 3.0, were used to grade the severity of side effects than four was 0% in the intensified group. Zero organ was 77.5%, one was 5%, two was 15%, three was 2.5% and more than four was 0% in the standard group. Documented infection, except febrile neutropenia, more than grade 3 was also analyzed until day100. 56.1% was detected in the intensified group and 40% in the standard group .Transplant-related mortality (TRM)p=0.09). No statistically significant differences were observed. TRM at 100 days was 34.2% (17.9-47.2%) for the intensified and 27.5% (12.2-40.1%) for the standard group (p=0.52).There was also no statistically significant difference  until day 30 post-transplantation was 17.1% (4.7-27.8%) for the intensified group and 5.0% (0-11.5%) for the standard group (fference .Relapsep=0.54). Moreover, non-relapse mortality occurred in some patients who had residual or relapsed diseases.  The cause of death is shown in Seven non-relapsed mortality (NRM) patients in the intensified group and 1 NRM patient in the standard group had relapse or residual disease at death. In total, 22 of 41(53.7%) patients in the intensified group and 13 of 40 (32.5%) in the standard group had residual diseases or relapses (data not shown). The disease is thought to be the biggest problem for HSCT even after the intensified regimen for those who did not achieve remission in non-remission hematological malignant diseases.30. So, PFS and OS must be relatively less evaluated in the intensified conditioning group in this retrospective analysis. If the disease backgrounds are the same, they must perhaps lead to less relapse and residual disease in the intensified group after allo- SCT. There are no other options to take against the non-remission hematological diseases before the SCT conditionings other than reinforcing the conditioning. For the purpose of shrinking the tumor burden, intensified conditioning is one of the meaningful options that can guarantee safety. Toxicity and safety of conditioning regimens were determined before SCT in each situation7,8. However, a few reports have stated that the intensified conditioning is better than the standard one14-21. Some authors also mention that different results were seen by the different doses and various combinations of chemotherapeutic drugs used in conditioning regimen2, 16, 17, 31, 32. Using novel conditionings in combination with new drugs have been developed showing better insight33, 34. In our analysis, ETRM was 17.1% for the intensified group and 5.0% for the standard group (p=0.09), which was not significantly different. TRM at 100 days was 34.2% for the intensified and 27.5% for the standard group (p=0.52). Meanwhile, no statistically significant difference was found in the non-remission cases. According to the results, we can try to modify the conditioning to get a better outcome by doing sequential and/or intensified chemotherapy, carefully considering the age, PS, organ function, etc. In general, to control the relapse and　refractory hematological malignant diseases following SCT, we can employ several methods: intensifying conditions, modifying the blood concentration of immunosuppressive drugs 20, inducing graft- versus- leukemia/lymphoma (GVLL) effects, using donor lymphocyte infusion (DLI) 35,36, selecting haploidentical siblings for donors 37,38, minimal residual disease and chimerism monitoring for pre-emptive administration35,39, etc. Recently, supportive therapy has progressed and, in cases of relapse, PS and organ function are often preserved and retransplantation could be considered 40,41. Regarding the sources of SCT, the use of cord blood or haploidentical sibling peripheral blood stem cells, which can be prepared easily and quickly, is gradually increasing and, by repeated SCT experiences, its use is becoming more familiar 37,38,42. Inducing GVHD/GVLL was also the choice for relapse disease by decreasing and ceasing the immunosuppressive drugs, tacrolimus, cyclosporine and DLI 20,35,36. Too much damage to the patient caused by conditioning regimen with cytotoxic drugs and radiation would narrow down the choices for relapse or residual disease. But, the outcome after relapse or residual disease after SCT are not usually favorable 35,36,39-41.Cure by means of single allo-SCT might be a simpler and better approach. Totally, intensified conditioning could be considered in cases of non-remission disease before HSCT. In this analysis, our results suggest that the OS could not be affected by intensified conditioning for the non-remission hematological malignant diseases. However, it was at least confirmed that TRM was not also significantly different at 30 days and 100 days for both groups. The main cause of death was relapse for both groups in our study. Even after intensifying the conditioning, relapse and residual disease were the most difficult problems we encountered. In this analysis, the patients’ backgrounds were a little different. Owing to the retrospective analysis, the doctors for each patient might have tended to choose intensified conditioning if the tumor burden was great. So, the intensified group was in a ‘worse disease’ status. In fact, we could confirm that 15 of 25 (60%) AML patients in the intensified group and 2 of 8 (25%) AML patients in the standard group had more than 30% blast cells in the bone marrow prior to beginning the conditioning regimen. Furthermore, in the lymphoma and ATLL patients, the disease status in 8 of 11 (72.8%) patients in the intensified group was more advanced than SD. The same result was also obtained in 11 of 23 (47.8%) patients in the standard group. One report stated that blast cells less than 26% in the bone marrow was one of the factors that contributed to better long-term survival in patients with leukemia not experiencing remission following allo-SCTOur study indicates that TRM of intensified regimens was not significantly different from that of standard regimens at 30 days and 100 days. The results also suggest that the intensified conditioning regimens can be employed just before HSCT to improve survival in patients with non-remission disease.


The treatment of traumatic low-degree vertebral compression fracture remains in a wide range between functional treatment, bracing, vertebroplasty, kyphoplasty, and even surgical fixation. The objective was to assess the innovation of instrumented kyphoplasty and to report the early and mid-term functional and radiological results. This study is a retrospective review of patients enrolled from 2012 to 2017. 104 consecutive endovertebral implantations of instrumented kyphoplasty were reviewed for the study. There were 56 women and 48 men. 93 of 104 patients were evaluated, of whom 27 were evaluated only by retrospective medical record review and 66 with follow-up visit. Clinical parameters were the pain rating scale (VAS) and the Oswestry score questionnaire. The radiological parameters were the vertebral kyphosis, vertebral height, lumbar lordosis, and adjacent disc degeneration . Statistical correlations between before/after surgery/last follow-up were performed. The average follow-up was 26.7 months (3 to 55). The average VAS decreased from 8.2 to 3.2 the day after surgery, allowing immediate standup. The average Oswestry score was 14.6 at follow-up. The average vertebral kyphosis decreased from 12.9° to 6.5° post-op and stabilized at 8.0° at the last follow-up, corresponding to 28% gain on vertebral height. The lumbar lordosis was restored (+6.6°). Adjacent disc degeneration increased by 1 UCLA grade in 17 patients (16.3%) at follow-up. The instrumented kyphoplasty in acute led to immediate and lasting pain relief, with no bracing or bed rest, short stay in hospital, and quick return to daily life including professional activities. The good clinical results were associated to a stable radiological restoration of the vertebral anatomy. Vertebral compression fractures (VCFs) represent a major health concern. Cases can be traumatic, osteoporotic, or metastatic. Their prevalence in patients over 50 years reaches 18% in Europe and 23% in the United States [Brace is a noninvasive option. It is a treatment of choice for patients with important comorbidities with a high-risk for anesthesia and surgery. Nevertheless, tolerance to the brace had been demonstrated to be lowered in elderly patients due  to the pMinimal invasive surgical treatment of VCFs by cement injection under imaging guidance has gained popularity since the first use of cement injected into a tumor cervical vertebra in France in 1984 . It has In our practice, patients had the choice between surgical procedure and orthopedic treatment after exposure to the advantages and disadvantages of each option.2. All patients had an A1 to A3 type of fracture in the Magerl classification diagnosed by a standing (if possible) X-ray anteroposterior and lateral and a preoperative CT scan [This study was a retrospective review of patients enrolled between May 2012 and January 2017. 104 consecutive patients with stable VCF, from the 2 hospitals in which our team worked, were all operated by instrumented kyphoplasty using SpineJack implants (Stryker) and polymethylmethacrylate cohesion cement (Stryker). These were 56 women and 48 men, with average age 54 years (18–83) with two frequency peaks around 40 and 65 years old . 51% of Clinical criteria were assessed by the visual analogue scale (VAS)  and the The radiological criteria were measured at each visit on the standing images  using the Carestream Vue Pacs® Version 11.4.0.1253 software. Local parameters of interest were vertebral kyphosis (VK), regional kyphosis (RK), and regional traumatic angulation (RTA) . The losp < 0.05.The statistical methodology included the use of descriptive statistics , parametric statistical tests (Fisher's test or the Wilcoxon), and nonparametric tests (Mann–Whitney). In particular, the normality of the variables was defined through the Shapiro–Wilk test of normality. Statistical significance was set at p < 0.05 compared to preoperatively); and 1.42 (SD = 1.87) at the last follow-up (p < 0.05 compared to 24 h). The average prefractural ODI score was 3.8 points (SD = 7.5) preoperatively; 26.6 points (SD = 23.95) at 6 weeks (p < 0.05 compared to preoperatively); and then 14.6 points (SD = 16.0) at the last follow-up (p > 0.05 compared to preoperatively). On average, the ODI score increased by 10.2 points (SD = 14.1) between the initial status (before the fracture) and the last follow-up. At last follow-up (mean 26.7 months and median 27 months), 53 patients (70.6%) had an ODI below 20 points. The return to daily activity was obtained between 7 and 10 days postoperatively, and the return to professional activity was obtained between 10 days and 7 months (average of 60 days). An adjacent vertebra fracture occurred in a 63-year-old osteoporotic woman who fell from her height 3 months after surgery.The average VAS was 8.15 (SD = 1.74) preoperatively; 3.21 (SD = 1.52) at 24 h . The average lumbar lordosis was 42.1° (SD = 9.7) preoperatively; 46.4° (SD = 9.2) at 24 h; and 48.7° (SD = 10.9) at the last follow-up. The variation of the lordosis and LL-PI mismatch are reported in Three patients (3.3%) had grade 1 preoperative disc degeneration at the superior adjacent level. No disc degeneration of the inferior adjacent disc was diagnosed preoperatively. At the last follow-up (mean 29 months), 17 patients had increased their degeneration disc by 1 grade. At the last follow-up, 20.3% had superior adjacent disc disease radiological signs. No degeneration sign of the inferior adjacent disc was observed .In total, no neurological or postoperative embolic complications have been recorded. 12 patients (11.6%) had cement leaks with no clinical consequences. 58.3% of the leaks were lateral. Our study reported good immediate clinical results and stable anatomical restoration over time in terms of local and regional radiological parameters. In spite of sagittal balance restoration, some extent of degenerative evolution of adjacent disc was noted, which did not impair the clinical results.The clinical results showed significant and immediate efficacy on the VAS within the first 24 h post-op. The only other study published using the SpineJack (Stryker) implants reported better immediate VAS improvement  at 48 h Our series reported an improvement of the VK from 12.9° to 6.5° in the same range compared to the 14.5° to 9.2° change in the series of Noriega et al.  with theThe restoration of vertebral height is another way to assess the quality of the reduction. We observed 46% immediate gain in anterior height in our series between the pre-op and post-op status. There were no data to refer to in the literature for instrumented KP. The study of Ates et al. regarding noninstrumented KP reported 11.8% improvement . VP and Kyphosis and vertebral height are involved in the forward sagittal imbalance, which is significantly associated with pain and loss of function . The impAssessment of stability of the radiological restoration in a longer term was another goal of our study. The secondary loss of VK improvement in our series was 1.5° between post-op and last follow-up (6.55° to 8.04°). In comparison, Hiwatashi et al.  reportedRegarding the progression of the disc degeneration, we observed in our series a mild evolution in the upper adjacent disc with 16.3% of cases worsening by 1 grade at follow-up on plain radiographs. Benneker et al. found that the radiological analysis is as reliable as MRI analysis with a strong correlation for the assessment of disc degeneration . In a seThis study has several limitations: having a noncomparative plan, the comparison is made only with the medical literature. It was retrospective and without a control group; hence, the evidence regarding the effectiveness of the surgery rather than the natural evolution of the healing process is limited. Nor did we consider any difference in disc degeneration between the lumbar and thoracic discs. Also, we did not distinguish subgroups of patients with high-energy and low-energy trauma which may implicate osteoporosis fracture.In conclusion, the instrumented kyphoplasty in acute provides immediate and lasting pain relief, with no bracing or bed rest and short stay in hospital. The good clinical results were associated to a stable radiological restoration of the vertebral anatomy.


Tuberous Sclerosis Complex (TSC) is a rare multisystem disorder. In 2012 diagnostic criteria for TSC were revised. However, data on the incidence of TSC are limited.Prospective, national surveillance study in Germany over a 2-year-period (03/2015–02/2017) using current revised criteria for TSC. Patients up to the age of 18 years with a new diagnosis of definite or possible TSC  were included. The aims of this study were 1) to generate up-to-date data on the incidence of definite or possible TSC, 2) to assess age at first diagnosis, and 3) to compare these data with previous epidemiologic data.In total, 86 patients met inclusion criteria (definite or possible TSC) with a median age at diagnosis of 6 months (range: 5 months before birth – 197 months of age). Among patients identified with features of TSC, 73.3% met criteria for definite diagnosis (median age: 7 months) and 26.7% met criteria for a possible diagnosis (median age: 3 months). 55.8% of patients were male. When excluding prenatally diagnosed patients, median age at diagnosis was 11 months with a range of 0 to 197 months. The 3 most common clinical features at diagnosis of TSC were central nervous system involvement in 73.3% patients (of these 95.2% experienced seizures), cutaneous involvement in 58.1% patients (with the most common lesion being hypomelanotic macules in 92%) and cardiac rhabdomyoma in half of the patients. Cardiac rhabdomyoma were detected by prenatal ultrasonography in 22.1% of patients. The presence of cardiac rhabdomyoma was associated with cardiac arrhythmias in 25.6%  in our cohort. The overall prevalence of seizure disorders was 69.8%. The annual incidence rate of TSC is estimated at a minimum of 1:17.785 live births. However correcting for underreporting, the estimated incidence rate of definite or possible TSC is approximately 1:6.760–1:13.520 live births in Germany.This is the first study that assessed prospectively the incidence rate of TSC in children and adolescents using the updated diagnostic criteria of 2012. This prospective surveillance study demonstrates a low age at first diagnosis (median: 6 months), likely due to antenatal detection of cardiac rhabdomyoma. Early diagnosis bears the potential for implementing effective therapies at an earlier stage.The online version of this article (10.1186/s13023-018-0870-y) contains supplementary material, which is available to authorized users. TSC1 or TSC2 genes (encoding for the protein Hamartin on chromosome 9q34 and Tuberin on chromosome 16q13 respectively) [Tuberous Sclerosis Complex (TSC) is a rare genetic neurocutaneous, multisystem disorder with a variable clinical phenotype –3. It isctively) . Benign ctively) . In 2012ctively) .Table 1DIn recent years large-scale data on the clinical and genetic characteristics have emerged, most importantly from the TOSCA (TuberOus SClerosis registry to increase disease Awareness) study. The TOSCA study is a large natural history study encompassing 2093 patients with TSC . In thisThe German Paediatric Surveillance Unit (ESPED) was founded in 1992 to generate incidence data and detailed clinical descriptions of rare, childhood-onset diseases in Germany requiring in-hospital treatment , 18. EleTo generate up-to-date data on the incidence of definite or possible TSC in Germany over a 2-year-period using current revised criteria for TSCTo assess age at first diagnosis, andTo compare our results with previous epidemiologic dataThe major aims of this prospective, national surveillance study were:This study was a prospective, national surveillance study conducted from March 1, 2015 to February 28, 2017 in Germany. The study was approved by the Institutional Ethics Review Board of Saarland, Germany (file no. 219/14). Patients up to the age of 18 years with a new diagnosis of definite or possible TSC [n = 349), all social pediatric centers (n = 120) and TSC centers (n = 18) in Germany, using the German Paediatric Surveillance Unit for Rare Diseases (ESPED) system [Electronic and postal questionnaires  system –19. Stat) system , 18.For classification of patients with a definite or possible diagnosis of TSC, we used the current Tuberous Sclerosis Consensus Conference updated diagnostic guidelines .Initially 150 cases were reported to ESPED, from which 135 patient questionnaires were received (response rate 90%). 40 questionnaires did not report TSC patients and 9 contained redundant datasets (double reporting) and were excluded. In total, 86 patients met inclusion criteria. Median age at diagnosis (definite or possible TSC) was 6 months (range: 5 months before birth – 197 months of age). Among patients identified with features of TSC, 73.3% (63/86) met criteria for definite diagnosis  and 26.7% (23/86) met criteria for a possible diagnosis . 55.8% (48/86) of patients were male. Age at diagnosis is shown in Fig. Based on our findings, the annual incidence rate of TSC (definite or possible TSC) is estimated at a minimum of 1:17.785 live births. However correcting for underreporting using data from previous ESPED analyses, the estimated incidence rate of definite or possible TSC is approximately 1:6.760–1:13.520 live births in Germany.Cardiac rhabdomyoma were detected by antenatal ultrasound in 22.1% of patients (19/86), leading to antenatal diagnosis of ‘possible TSC’. One patient had concomitant cerebral abnormalities (no other characteristics were reported).The most common clinical feature at diagnosis of TSC was central nervous system (CNS) involvement in 73.3% patients (63/86), of these 95.2% (60/63) experienced seizures. Hence, the overall prevalence of seizure disorders in our cohort was 69.8% (60/86). Cutaneous involvement was seen in 58.1% patients (50/86); with the most common lesion being hypomelanotic macules in 92% (46/50). Cardiac rhabdomyoma manifested in half of the patients (43/86), 25.6% of these (11/43) with cardiac arrhythmia.The spectrum of TSC manifestations other than cardiac, cutaneous and CNS involvement was heterogeneous Fig. . ResultsTests used to establish the diagnosis are detailed in Fig. TSC1: 21.7% (10/46); TSC2: 58.7% (27/46); no mutation identified (NMI) 19.6% (9/46)). Of note, two patients with TSC2 mutation also had a PKD1 mutation (contiguous gene syndrome). A family history of TSC was found in 13 of 86 patients (15.1%).Results from genetic testing were available in 53.5% patients (46/86): . Interestingly, our incidence data were similar to previous reports, where incidence rates in adults were calculated by employing prevalence data analysis . Our datnce data . These tnce data .Davis et al. in which cardiac rhabdomyoma were the most common initial presenting feature of TSC [Bader et al. the mean gestational age at diagnosis of cardiac rhabdomyoma was 28.4 ± 6.0 weeks  [TSC is a very heterogeneous disorder both with regard to age-related expression and variability of clinical manifestations . Of notee of TSC . Comparee of TSC , mandatie of TSC . Of notee of TSC . Routinee 19–37) . Howevere 19–37) . FurtherDavis et al. demonstrating a prevalence of tubers or cortical dysplasia of 94% in their cohort [CNS involvement was the most common clinical symptom at diagnosis in our study. These findings are congruent with the results from r cohort  as well r cohort . The oveWith new treatment options (e.g. mTOR inhibitors) for a variety of TSC manifestations   and for This is to our knowledge the first population-based estimation of definite or possible TSC incidence in children using current diagnostic criteria, thus providing the medical community with a robust estimate of the incidence of TSC in children. In contrast to our dataset, other studies have reported prevalence rates –15 and/oAdditional file 1:TSC Questionnaire ESPED Germany. (PDF 75 kb)


According to several studies in population of high-income countries (HIC), patients with Type 2 diabetes mellitus (DM) have a considerably higher risk of cardiovascular morbidity and mortality. However, it is not clear if the magnitude of this association can be widespread in other populations. The objective of this study was to determine the independent association between Type 2 DM and first cardiovascular event in Colombian Caribbean poor population with no records of previous cardiovascular events reported.Kaplan–Meier curves were constructed for the cardiovascular events related endpoints and matched Cox-regression analysis to estimate associations of a history of Type 2 DM with cardiovascular outcomes during 1.5 years of follow-up. A formal sensitivity analysis using The Breslow-Day and Tarone Homogeneity tests was conducted.We retrospectively reviewed the individual records from the hospitalizations database of 64,668 patients of cardiovascular risk management program from July 2014 to December 2015. We used a propensity score matching cohort analysis for this study. The p = 0.000), AMI  and stroke . Hazard ratios (95% CIs) for the association of Type 2 DM with all-cause mortality, cardiovascular mortality and all-cause hospitalization were 1.36 , 1.52 , and 1.20 , respectively.Out of 56,351 patients with no previous cardiovascular events records, 19,368 (34.4%) patients were found to suffer Type 2 DM. Using propensity scores for Type 2 DM, we gathered a cohort of 18,449 pairs of patients with and without Type 2 DM who were balanced on 22 baseline characteristics. A first cardiovascular event occurred in 650 (3.5%) and 403 (2.1%) matched patients with and without Type 2 DM, respectively, during 1.5 years of follow-up. Type 2 DM was associated with first cardiovascular event (HR 1.69; 95% CI 1.43–2.00; Type 2 DM resulted to be a significant independent risk factor for first cardiovascular event in Colombian Caribbean poor population with no previous records of cardiovascular events. Diabetes mellitus (DM) is responsible for high global mortality rates and high disability burden  and is aIn the world, 80% of cases of noncommunicable diseases (NCDs) occur in LMICs . Most coThe objective of this study was to examine the association between Type 2 DM with first cardiovascular event in a cohort of type 2 DM Colombian Caribbean poor population with no previous records of cardiovascular events compared with a paired matched cohort of patients with no presence of DM.In 2015 Colombia had 48.2 million people. 10.3 million of them live in the Caribbean Region. For this study, patients from a Colombian public health insurance company were selected. By 2015, it had 1.2 million insured people from poor households of the Colombian Caribbean region (11.6% of the population in the Caribbean region). 49% are male, sex ratio 1:1 and 54% are under 30 years old, which indicates a representative and very similar population structure to the Colombian Caribbean region .The insurance company have a cardiovascular risk management program, which performs screening of cardiovascular risk factors and the detection of cardiovascular events in the affiliated population. Screened patients who reported presence of risk factors were enrolled in the cardiovascular risk management program, which carry out a plan of follow-up activities according to the stratification of cardiovascular risk from the Framingham Risk Score.Sociodemographic and clinical characteristics of enrolled patients from the program are registered in a private platform of the health insurance company. For the current investigation, we had access to the individual records of service provision registered between July 2014 and December 2015. In 2015, there were 64,668 (66.5% women) patients enrolled in the program. More than 90% of them were over 45 years old.Patients with type 2 DM were identified through at least once of the following characteristics: having ICD − 10 diagnosis code, having a personal reported history of DM, having glycosylated hemoglobin levels> 6.5% at least once in records and those under anti-diabetic treatment. From the identified patients, we excluded the ones with a personal history of previous cardiovascular events, and diabetics younger than 30 years under treatment with insulin and without oral anti-diabetics.For the current analysis, the main outcome were first CVD events related endpoints: such as acute myocardial infarction, angina pectoris, acute stroke, mortality due to CVD, and hospitalization due to CVD during 1.5 years of follow-up . CVD events were confirmed by ICD-10 diagnosis CVD events related endpoints from the cardiovascular risk management program hospitalizations database. Follow-up was censored with presence of cardiovascular events, death, or the end of the study, whichever occurred first.For the current investigation, we had access to hospitalizations records between July 2014 and December 2015, which were previously verified by the internal auditors of the health insurance company. The exact date of death of the patients was not available. So the date of hospitalization due to cardiovascular events or any other date was considered and imputed as the date of death in patients registered as “deceased” in the database of the program whose date of hospitalization was after the date of the last monitoring control observed. In the patients registered as “deceased” who did not have a record of hospitalizations, the median date between the date of the last control observed and the expected date of the next control was considered as the date of death.P value of less 0.20 was defined for selecting variables for entry into the final model. Selected variables were as follows: age, sex, smoking, physical activity, hypertension, obesity, hyperlipidemia, chronic kidney disease (CKD), hypertension control, Angiotensin-Converting Enzyme Inhibitors (ACEI)-Angiotensin-Receptor Blocker (ARB), calcium-antagonists, statins, antiplatelet and number of anti-hypertensive drugs. By using these covariates, a propensity score was calculated for each patient. Finally, each patient who underwent Type 2 DM was matched to one patient who underwent no Type 2 DM with the closest propensity score. The maximum difference of propensity score for a match was less than 0.03. To ensure that the post-match comparisons between patients with and without diabetes were not affected by the small sample size of the matched cohort, we assembled a pre-match cohort of the same sample size as that of the matched cohort. This was done by first identifying the 18,449 patients with diabetes in the matched cohort. Then, we identified a random sample of 18,449 patients without diabetes from the entire pre-match sample of 36,898 patients without diabetes. Finally, we linked these two data sets, thus assembling a cohort of 18,449 pairs of patients with and without diabetes.We used the propensity score method and performed 1:1 nearest-neighbor matching without replacement due to imbalances in baseline characteristics between patients with and without Type 2 DM. We used a non-parsimonious multivariable logistic regression model to estimate propensity scores for Type 2 DM. In the model, Type 2 DM was the dependent variable and clinically relevant baseline characteristics were used as covariates. Then a Pearson Chi-square and Wilcoxon Rank-Sum tests for the pre-match comparison and paired sample t-test for the post-match comparisons of baseline covariates between patients with and without DM. The Kaplan–Meier curves were constructed for the cardiovascular events related endpoints and matched Cox-regression analysis to estimate associations of a history of Type 2 DM with cardiovascular outcomes during 1.5 years of follow-up. Log-minus-log scale risk plots were used to check proportional hazards assumptions. A formal sensitivity analysis using The Breslow-Day and Tarone Homogeneity tests was conducted. All statistical analyses were conducted using the Stata version 14.2 software  and R version 3.4.3 . P value < 0.05 was considered statistically significant for all tests.Baseline characteristics were presented through absolute and relative frequencies and means with standard deviations. We used All 56,351 patients included in the study are from a low socioeconomic level. We could not access to information on educational level, civil status and other sociodemographic characteristics due to restrictions of the insurance company. After identification, 19,368 (34.4%) patients were identified through the Type 2 DM records as follow; 14,131 (73%) by the ICD-10 diagnosis of DM, 323 (1.7%) by a personal history of DM, 2334 (12.1%) by glycosylated hemoglobin levels> 6.5% at least once and 2579 (13.3%) by the reported treatment with anti-diabetic medicines. Patients were significantly younger in the personal history of Type 2 DM (−) group compared with the personal history of Type 2 DM (+) group (63.6 ± 13.1 years old versus 65.6 ± 13.6 years old); the number of anti-hypertensive drugs in the personal history of Type 2 DM (+) group is lower compared with the personal history of Type 2 DM (−) group (0.69 ± 0.45 versus 0.72 ± 0.44). 36.7% achieved good metabolic control (glycosylated hemoglobin < 7%).The incidence rate of first cardiovascular event in > 65 years old was 22.9/1000 person – year (95% CI 21.0–25.0) (Incidence Rate Ratio (IRR) 3.15 when ≥ 65 years old group was compared with < 65 years old group, 95% IC 2.65–3.75). The incidence rate of first acute myocardial infarction or angina pectoris event (AMI) in > 65 years old was 13.9/1000 person – year (95% CI 12.4–15.5) (IRR) 2.63 when ≥ 65 years old group was compared with < 65 years old group, 95% IC 2.14–3.26). The incidence rate stroke in > 65 years old was 9.31/1000 person – year (95% CI 8.13–10.67) .There were several parameters of baseline characteristics statistically higher in the family history of DM (+) group, including the percentage of female gender (68.07% vs. 65.33%), hypertension control (90.74% vs. 89.28%), use of antiplatelet (41.59% vs. 37.12%) and use of statins (52.05% vs. 44.54%). The incidence rate of first cardiovascular event in man was 20.9/1000 person – year (95% CI 18.6–23.5) . The incidence rate of AMI in man was 14.2/1000 person – year (95% CI 13.3–16.3) . The incidence rate of stroke in man was 6.95/1000 person – year (95% CI 5.67–8.51) .Among 19,368 subjects with a personal history of Type 2 DM (+) group, 667 (3.4%) subjects had a first cardiovascular event before propensity score matching. Among 18,449 subjects with a personal history of Type 2 DM (+) group, 650 (3.5%) subjects had a first cardiovascular event after propensity score matching. The incidence rate of first cardiovascular event was 22.2/1000 person – year (95% CI 19.9–24.7) (IRR 1.86 when Type 2 DM (+) group was compared with Type 2 DM (−) group, 95% IC 1.65–2.15). The incidence rate of AMI was 14.4/1000 person – year (95% CI 12.6–16.4) (IRR 2.00 when Type 2 DM (+) group was compared with Type 2 DM (−) group, 95% IC 1.67–2.39). The incidence rate of stroke was 8.05/1000 person – year (95% CI 6.73–9.63) (IRR 1.66 when Type 2 DM (+) group was compared with Type 2 DM (−) group, 95% IC 1.31–2.11).Among 19,368 subjects with a personal history of Type 2 DM (+) group, 3418 (17.4%) subjects were under treatment of human or analogous insulin. Of these, 213 (6.2%) subjects had a first cardiovascular event before propensity score matching. The incidence rate of first cardiovascular event was 41.01/1000 person – year (95% CI 34.08–49.36) (IRR 2.05 when Type 2 DM (+) group under human or analogous insulin treatment was compared with Type 2 DM (+) group without treatment with human or analogous insulin, 95% IC 1.73–2.42). The incidence rate AMI was 27.4/1000 person – year (95% CI 21.90–34.44) (IRR 2.03 when Type 2 DM (+) group under human or analogous insulin treatment was compared with Type 2 DM (+) group without treatment with human or analogous insulin, 95% IC 1.65–2.50). The incidence rate of stroke was 14.64/1000 person – year (95% CI 10.74–19.97) (IRR 2.13 when Type 2 DM (+) group under human or analogous insulin treatment was compared with Type 2 DM (+) group without treatment with human or analogous insulin, 95% IC 1.60–2.80).To explore this imbalance, we illustrate a histogram of propensity score distribution for groups with and without personal history Type 2 DM before Fig. n = 18,449) and without  Type 2 DM are shown in Table According to the propensity score matching 1:1 shown in Table In the group with and without a personal history of DM type 2, the first CVD event occurred in 650 (3.5%) and 403 (2.1%) patients, respectively (incidence rate 22.1/1000 person – year (95% IC 19.8–24.6) and HR 1.69; 95% CI 1.43–2.00; Fig. p = 0.000; Fig. p = 0.001; Fig. In the group with and without a personal history of DM type 2, the first acute myocardial infarction or angina pectoris event (AMI) occurred in 424 2.3%) and 254 (1.3%) patients, respectively (incidence rate 14.4 person -year (95% CI 12.6–16.5); HR 1.79; 95% CI 1.45–2.20; % and 254Significant unadjusted associations were estimated between the personal history of DM type 2 and several outcomes among the pre-match cohort. Among the 36,898 balanced cohorts, type 2 DM was associated with an increased risk of hospitalization from any cause and association with cardiovascular death, stroke death, and death from all causes Table .Table 5EFramingham Heart Study made it possible to identify DM as an important cardiovascular risk factor, mainly in women [Framingham Heart Study for stroke, women with DM had a higher incidence than men did with DM [The data from the in women . To the in women , 17. In in women . In our Framingham Heart Study, an increased mortality due to CVD in patients with DM compared with patients without DM was reported [The findings of the current analysis demonstrate that in patients without a personal history of previous cardiovascular events, a history of Type 2 DM was associated with an increased risk of a first cardiovascular event, which was primarily driven by an increase in AMI. Type 2 DM was also associated with risk of all-cause hospitalization, cardiovascular death, stroke death and all-cause death, but had no independent association with cardiovascular hospitalization. In the reported . In the reported . In the reported . These rreported . In randreported . In the reported . In the In Colombia, DM is among the first 10 causes of general mortality and within the first 20 causes of years of healthy life lost (YHLL) in a population ≥ 45 years  and the Care of a patient with DM requires a multifactorial approach from the cardiovascular risk management program. All patients are at risk of developing vascular complications of DM, and these risks represents ultimately result in a doubled risk of mortality in patients with DM. Above and beyond targeted interventions, we now know that strict multifactorial interventions can result in a clinically significant reduction in both mortality and cardiovascular disease. These conclusions provide significant information on the importance of Type 2 DM in increasing the risk of first cardiovascular events in the poor population of the Colombian Caribbean region and the importance of an adequate diagnosis and treatment of Type 2 DM. However, evidence that an adequate metabolic control reduces the rates of cardiovascular events and death is not clear, although a discrete cardiovascular benefit may be observed after a prolonged follow-up period –24. EvidOur study has several strengths and limitations. More than 35 thousand of patients were included and, in spite of the nearly 1000 patients with type 2 DM lost, there were no differences in the incidences and incidence rates between the groups after propensity matching. We tried that the possibility of residual confounding, by the measured covariates and confounding by the unmeasured covariates that explain the associations between Type 2 DM and first cardiovascular event, were unlikely, considering that our patients were matched in 22 baseline characteristics and the conclusions of our analysis of sensitivity suggest that the association between Type 2 DM and a first cardiovascular event was quite insensitive to an unmeasured binary confounder.Within limitations, we faced restrictions to access to sociodemographic data due to restrictions from the insurance company. In order to identify the diagnosis of cardiovascular outcomes we used the records of previous cardiovascular events and the ICD-10 hospital discharge code. However, we could not have data on confirmatory tests.On the other hand, the exact date of death of patients and date of enrollment on the cardiovascular risk management program was not available.We did not have access to information related to the treatment of cardiovascular events such as medications, thrombolysis, percutaneous coronary intervention (PCI), coronary artery bypass surgery (CABG), etc. In conclusion, in the poor population of the Colombian Caribbean without a personal history of cardiovascular events receiving standard therapy, DM produces cardiovascular outcomes. It is not known if a more aggressive screening of DM, DM education at every level, quality care registries and taxes on sugary drinks can reduce the risk of a first cardiovascular event in these patients and should be determined prospectively in future studies in Latin America.


Vibrio cholerae O1 and O139), whole-cell oral cholera vaccine  in healthy adults (n = 100) and children (n = 100) in a cholera endemic area  to fulfill post-licensure regulatory requirements and post-World Health Organization (WHO) prequalification commitments. Safety and reactogenicity were assessed, and seroconversion rates  were determined 14 days after each vaccine dose. No serious adverse events were reported during the study. Commonly reported solicited adverse events were headache and general ill feeling. Seroconversion rates after the first and second dose in adults were 67.7% and 55.2%, respectively, against O1 Inaba; 47.9% and 45.8% against O1 Ogawa; and 19.8% and 20.8% against O139. In children, seroconversion rates after the first and second dose were 80.2% and 68.8%, respectively, against O1 Inaba; 72.9% and 67.7% against O1 Ogawa; and 26.0% and 18.8% against O139. The geometric mean titers against O1 Inaba, O1 Ogawa, and O139 in both adults and children were significantly higher after each vaccine dose compared to baseline titers . The seroconversion rates for O1 Inaba, O1 Ogawa, and O139 in both age groups were similar to those in previous studies with the vaccine. In conclusion, the killed, bivalent, whole-cell oral cholera vaccine has a good safety and reactogenicity profile, and is immunogenic in healthy adults and children.This open-label study assessed the safety and immunogenicity of two doses (14 days apart) of an indigenously manufactured, killed, bivalent (Trial Registration: ClinicalTrials.gov NCT00760825; CTRI/2012/01/002354. Cholera continues to be a significant public health problem in many developing countries; globally, there are an estimated 1.3–4 million cases and 21,000–143,000 associated deaths annually.) and general ill feeling (28% [n = 28]). Most of the AEs were mild to moderate in intensity. Only one adult participant reported an AE  that was of severe intensity and which resolved by the following day.Among children, 13 (13%) reported solicited AEs during the study. The most commonly reported solicited AEs were general ill feeling (6% [n = 6]) and headache (5% [n = 5]). One child experienced severe headache and general ill feeling two days after the second dose (on Day 16) which resolved the following day.The most commonly reported unsolicited AEs among adults were headache (9% [n = 9]), abdominal pain or cramps (7% [n = 7]) and general ill feeling (6% [n = 6]), and the most commonly reported unsolicited AEs among children were fever (6% [n = 6]), abdominal pain or cramps (4% [n = 4]) and cough (4% [n = 4]). All the unsolicited AEs reported were assessed to be unrelated or unlikely to be related to the study vaccine.The seroconversion rate after each dose of the vaccine is summarized in V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 in adult participants ranged from 2.5 to 5120, 2.5 to 10240 and 2.5 to 960, respectively, and those in children participants ranged from 2.5 to 10240, 2.5 to 20480 and 2.5 to 60, respectively.The baseline vibriocidal antibody titers for V. cholerae O1 Inaba and relatively fewer number of participants seroconverted for V. cholerae O139. However, the difference in seroconversion for O1 and O139 was not tested for statistical significance.In both adult and child cohorts, the highest number of participants who seroconverted was seen for V. cholerae O1 Inaba and V. cholerae O1 Ogawa were higher in children than in adults . However, the difference in seroconversion rates for adults and children was not tested for statistical significance. Of note, adults and children with baseline antibody titers ≤ 80 V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 was observed after the first dose compared to that after the second vaccine dose (except for seroconversion rates in adults for V. cholerae O139) . Similar trends were observed in children age subgroups 1–4 years and 5–17 years, except for the seroconversion rate in children aged 1–4 years for V. cholerae O1 Ogawa . However, the relatively lower seroconversion rate following the second dose compared to that after the first dose was not observed in adults and children with baseline antibody titers ≤ 80, except for the seroconversion rate in children for V. cholerae O139 .A higher seroconversion rate in adults and children for V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 were significantly increased 14 days after both dose 1 and dose 2 of the vaccine, relative to baseline (P < 0.001). Again, relatively higher GMTs in adults and children for V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 were observed after first dose compared to that after the second vaccine dose. The GMT results are shown in  A similar trend of relatively lower GMTs after the second dose compared to that after the first dose was observed in children in the age subgroups 1–4 years and 5–17 years Figs  and 5.In both adults and children, vibriocidal GMTs for V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 after one and two doses of the vaccine is shown in  GMF-rise in titers for all the three serotypes were higher in children than in adults. However, the difference in GMF-rise for adults and children was not tested for statistical significance. In both adults and children, the highest GMF-rise in titers was seen with V. cholerae O1 Inaba . The lowest GMR-rise in titers was seen with V. cholerae O139 .The GMF-rise in titres in adults and children for The present study was conducted in India to evaluate the safety and immunogenicity of the fully scaled-up, indigenously manufactured, killed, bivalent (O1 and O139) whole-cell OCV in healthy adults and children residing in a cholera endemic area . Similar to the previous studies conducted in Kolkata (a phase II study in 2005 and a phase III study in 2007), the OCV V. cholerae O1 Inaba and O1 Ogawa in both adults and children. These results show that the vibriocidal responses to OCV are inversely related to serum vibriocidal antibody titers at baseline. However, even in adults and children with baseline antibody titers ≤ 80, 9% to 7% of the participants did not achieve seroconversion. It has previously been postulated that other factors such age, nutrient deficiency or the lack of response to B cell independent carbohydrate antigens may play a role in the lower vibriocidal responses.[The present study also demonstrated that the vaccine was immunogenic in a cholera endemic population, where participants have high pre-existing vibriocidal antibody titers. Among participants with low baseline antibody titers ≤ 80, higher rates of seroconversion (over 90%) were observed for esponses., 14V. cholerae O1 Inaba and lowest for V. cholerae O139. Even among participants with baseline antibody titers ≤ 80, the seroconversion rate was low for V. cholerae O139. The lower immune response to O139 may be related to the sensitivity of the assay used; as previously suggested, the highly diluted complement, used in the vibriocidal assay for V. cholera O1, may not be sufficient to facilitate killing of V. cholerae O139 which is capsular.[For both adults and children, the seroconversion rate was highest for capsular., 15, 16V. cholerae O1 Inaba, V. cholerae O1 Ogawa and V. cholerae O139 were observed after the first dose compared to that after the second dose. The lack of increase in immune responses after the second dose has also been observed with an extended 28-day interval between the two doses in another study.[Vibrio cholera infection given that the study was conducted in a cholera endemic area. It is also possible that the first dose may act like a booster in some participants given the high cholera endemicity in the region.[Similar to previous studies with the OCV in cholera-endemic areas,, 10 higher study. The higher study., 17, 18 er study., 17 The .[V. cholerae O139 in adults after the second dose, with higher seroconversion rates in present study compared to that undertaken in 2007 . The reason for this difference may be due to optimization of the vibriocidal assay since the earlier study, and the use of lower complement concentrations.[The immunogenicity of OCV in this study was similar to that of other studies with the vaccine conducted in cholera endemic areas ., 6 The b. and 186 trations.As with the previous studies with whole-cell OCV, this study conducted with the fully scaled-up, indigenously manufactured vaccine demonstrated that a two-dose regimen (14 days apart) of the vaccine was well tolerated and immunogenic in adults and children aged at least 1 year.S1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


The extent of molecular diversity parameters across three rice species was compared using large germplasm collection genotyped with genomewide SNPs and SNPs that fell within selective sweep regions.Oryza glaberrima Steud.) as compared to its wild progenitor (O. barthii A. Chev.) and to Asian rice (O. sativa L.). Here, we characterized a large collection of African rice and compared its molecular diversity indices and population structure with the two other species using genomewide single nucleotide polymorphisms (SNPs) and SNPs that mapped within selective sweeps. A total of 3245 samples representing African rice (2358), Asian rice (772) and O. barthii (115) were genotyped with 26,073 physically mapped SNPs. Using all SNPs, the level of marker polymorphism, average genetic distance and nucleotide diversity in African rice accounted for 59.1%, 63.2% and 37.1% of that of O. barthii, respectively. SNP polymorphism and overall nucleotide diversity of the African rice accounted for 20.1–32.1 and 16.3–37.3% of that of the Asian rice, respectively. We identified 780 SNPs that fell within 37 candidate selective sweeps in African rice, which were distributed across all 12 rice chromosomes. Nucleotide diversity of the African rice estimated from the 780 SNPs was 8.3 × 10−4, which is not only 20-fold smaller than the value estimated from all genomewide SNPs (π = 1.6 × 10−2), but also accounted for just 4.1%, 0.9% and 2.1% of that of O. barthii, lowland Asian rice and upland Asian rice, respectively. The genotype data generated for a large collection of rice accessions conserved at the AfricaRice genebank will be highly useful for the global rice community and promote germplasm use.Previous studies conducted on limited number of accessions have reported very low genetic variation in African rice (The online version of this article (10.1007/s00122-018-3268-2) contains supplementary material, which is available to authorized users. Oryza L.) is one of the top three food crops in the world, ranked second in total area harvested and production per hectare and third in total global production after maize and wheat . The genus Oryza consists of 27 species and 11 genome types , BB (O. punctata), CC (O. eichingeri), FF (O. brachyantha) and BBCC (O. schweinfurthiana) genome species , 85% to Asian rice (O. sativa) and 1% of the other wild species.Rice  sweeps, which refer to a reduction in nucleotide diversity near advantageous mutations in useful genes, leave distinct signatures in genomes; this enables the detection of loci that have undergone positive selection  identify genomic regions that have undergone selective sweeps in African rice and examined if those selective sweeps showed greater reduction in nucleotide diversity in African rice as compared with its wild relative O. barthii and Asian rice and (2) compare the extent of genetic relatedness and population structure of the three rice species using genomewide SNPs.A wide range of statistical methods have been used to identify traces of intra-species selective sweeps by detecting regions of reduced genetic variation, which have undergone a selective sweep , African rice (2358 samples) and Asian rice (772 samples). About 92% of the African rice  of which 26,073 SNPs (Dataset-1) were physically mapped on to the 12 rice chromosomes and had two alleles each irrespective of their minor allele frequency  conserved at the AfricaRice genebank (Supplementary Table S1), which represent θS), nucleotide diversity (θπ) and Tajima’s D test statistic  a minicore of 350 accessions .Hapmap input files from TASSEL v.5.2.48 were exported to PHYLIP interleaved format, which were then converted to both MEGA X  and principal component analysis .OmegaPlus v.3.0.2 , lowland O. sativa (Dataset-5) and upland O. sativa (Dataset-6) were also used to compute nucleotide diversity in MEGA X , D value computed from the 26,073 SNPs in Dataset-1 was positive in the wild O. barthii (0.219), but negative in the cultivated African (− 1.381) and Asian rice (− 0.257 for lowland and − 1.513 for upland O. sativa). The negative Tajima’s D values observed in the two cultivated rice species are indicative of positive selection and reject the null hypothesis that all mutations are selectively neutral. Using OmegaPlus, we identified a total of 37 candidate selective sweep regions in African rice . Selective sweeps increase the frequency of beneficial alleles and surrounding variants and may eventually lead to fixation, and recombination and mutation that introduce new alleles are rare , which are evident in Supplementary Table S2.Table D value in O. barthii supports the null hypothesis of selective neutrality, results of OmegaPlus revealed 36 selective sweeps in this wild species of which six regions  were shared with those detected in African rice . Overall, we identified a total 1353 SNPs, 1426 SNPs and 1421 SNPs that fell within the selective sweeps detected in O. barthii, lowland and upland Asian rice, respectively, which were used for computing molecular diversity indices (see below).Selective sweeps were more common in Asian rice than in African rice Table . Each se5–28.6 × 105) were observed in 850 kb interval on chromosome 9 . This region harbored clusters of 87 protein coding genes (Supplementary Table S4), including DEEPER ROOTING 1 (DRO1), that play role for adapting to the dry sub-Saharan region and DELAYED SEED GERMINATION 1 (OsDSG1) that prevent germination when there is insufficient moisture in the soil and under storage conditions. The role of other candidate genes with known function has been summarized in Supplementary Table S3 and described in detail in the discussion section.To gain insight into possible roles of each selective sweep in African rice, we compiled a list of 901 protein coding candidate genes found within the 37 selected regions (Supplementary Table S4). There were two to ninety-two genes per selective sweep region of which some had known functions, descriptions of which can be found in Supplementary Table S3. As shown in the Manhattan plot in Fig. O. barthii, but 64.2% and 46.3% smaller than those of the lowland and upland Asian rice, respectively (Table O. barthii (0.041), 29.6% of the lowland and 38.4% of the upland Asian rice. Nucleotide diversity (π) in African rice was 0.016, which accounted for 37.1%, 16.7% and 37.3% of the values observed in O. barthii (0.044), lowland (0.097) and upland (0.043) Asian rice, respectively. The number of segregating sites, proportion of polymorphic sites and ϴ and π values computed from the 23,079 SNPs in Dataset-2 that were polymorphic across all 3245 samples at a MAF ≥ 0.01 were similar to that of Dataset-1.Of the 26,073 SNPs in Dataset-1, the number of segregating sites (6843) and proportion of polymorphic sites (0.262) in African rice were nearly 20% greater than those of −4, which accounted for just 4.1% of that of O. barthii, 0.9% and 2.1% of the lowland and upland Asian rice, respectively (Table O. barthii (Dataset-4) and lowland Asian rice (Dataset-5) accounted for 55.2% and 34.4% of that of O. barthii and lowland Asian rice, respectively, which are even higher than the values obtained from the entire 26,073 SNPs in Dataset-1. These results demonstrate a more severe reduction in genetic variation in African rice due to selective sweeps detected in this species than the selective sweeps detected in the other two species. The nucleotide diversity of African rice as calculated from SNPs in selective sweeps identified in upland Asian rice (Dataset-6) was sevenfold greater than the value obtained in Dataset-3, but smaller than that of Dataset-1. The latter is not surprising due to not only much smaller Tajima D value observed in upland than the lowland Asian rice  as compared to just twofold in O. barthii (π = 4.4 × 10−2 in Dataset-1 vs. 2.0 × 10−2 in Dataset-3). On the contrary, the changes in nucleotide diversity in lowland and upland Asian rice were very minimal irrespective of the datasets; the only exceptions were Dataset-5 in the lowland Asian rice and Dataset-6 in the upland Asian rice that showed about from two to threefold smaller nucleotide diversity values as compared to the whole 26,073 SNPs.Using genotype data of the 780 SNPs (Dataset-3) that fell within the 37 selective sweeps detected in African rice, we compared the changes in nucleotide diversity across the three species. Nucleotide diversity in African rice was 8.3 × 10N = 115, 163, 350, 983 and 1375) using the 26,073 SNPs. Nucleotide diversity of African rice computed from these five smaller sample sizes accounted from 30.5 to 45.9% of that of O. barthii, from 13.5 to 20.6% of that of lowland Asian rice and from 30.1 to 46.2% of that of upland Asian rice . The first group consisted of all samples belonging to the African rice and O. barthii. The second and third groups consisted of all Asian rice samples adapted to the lowland (which are primarily indica) and the upland (primarily japonica) ecologies in Africa, as summarized in Supplementary Table S1. The first two principal components from PCA performed across all three species accounted for 84.7% of the molecular variation observed across the 3245 samples. A plot of PC1 (74.3%) and PC2 (10.4%) showed clear population structure in the same way as the neighbor-joining analysis . Nearly 56% of the pairs of samples differed by just < 5% of the scored alleles. All pairs of African rice samples, 50.2% of the O. barthii, 54.1% in lowland Asian rice and 34.0% in upland Asian rice . In African rice, the genetic distance values for about 78% and 12% of the pairs of accessions varied from 0.101 to 0.200 and from 0.201 to 0.300, respectively, with none of the pairs having a genetic distance exceeding 0.308. On the contrary, approximately 22%, 50% and 24% of the pairs of O. barthii accessions had genetic distance values ranging from 0.101 to 0.200, from 0.201 to 0.300 and from 0.301 to 0.400, respectively  was 10.9% (2840 SNPs) in African rice, 18.4% (4807 SNPs) in O. barthii (0.241) and lowland Asian rice (0.190), respectively, but it was 12.6% greater than that of upland Asian rice (0.135) (Supplementary Table S7). The phylogenetic tree constructed from the genetic distance matrix computed from 3484 SNPs that were polymorphic in both African rice and O. barthii showed three major groups, with the African rice samples partitioned into six subgroups . Plots of PC1 and PC2 from PCA  showed similar pattern as the cluster analysis. Detailed results of 2179 of the 2358 (94.4%) African rice samples have been presented in our previous study  into four hierarchical levels corresponding to African rice, O. barthii, lowland Asian rice and upland Asian rice revealed that differences among groups accounted for 92.4% of the total variation  accounted for 63.1% and 80.0% of the average distance observed in rthii 0.21 and lowO. barthii, which are consistent with previous studies irrespective of marker types and density , whereas the other accessions sampled outside the domestication center formed three additional groups. In the current study, we found six groups of African rice and that the group membership of the O. barthii samples generally agrees with their country of origin but not ecology. However, nearly 40% of the O. barthii accessions were clustered together with African rice accessions, which differed between the current study and other studies  the identification of selective sweeps, (2) the genetic diversity ratio of the wild and cultivated species and (3) the identification of loci that had undergone extreme genetic differentiation . Some of the candidate genes that fell within the selective sweep region on chromosome 9 with the highest Omega scores were a Jasmonate ZIM-domain protein that induces resistance to bacterial blight  and harbors 5 candidate genes . The latter includes an aminotransferase-like enzyme, which is involved in a number of metabolic activities, including abiotic stress in rice  when the analyses were conducted on the genotype data of 780 SNPs that fell within 37 selective sweep regions identified in this species. These regions contained various annotated genes other than the well-known domestication genes whose functions provide additional clues as to how domestication proceeded from O. barthii to cultivated African rice.The present study was conducted on a large collection of genotypes representing the three AA-genome MNN conceived, designed and supervised the experiments, secured funding and partly drafted the paper; AG and SBK were responsible for sample preparation, DNA extraction and/or compilation of passport information; NA conducted the selective sweep analyses; KS conducted most analyses and wrote most part of the paper; DZ and PP contributed to and edited the paper. All authors read and approved the paper.Supplementary material 1 (7z 9 kb)Below is the link to the electronic supplementary material.


Tobacco is a leading preventable cause of non-communicable diseases (NCDs). Studies characterizing the prevalence of tobacco use in low-income countries are lacking. This study describes the prevalence of tobacco use in Burkina Faso and its associated factors.Data from the 2013 Burkina Faso World Health Organization (WHO) Stepwise approach to Surveillance (STEPS) were analyzed. The prevalence of any tobacco product use, cigarette smoking, and other tobacco use was calculated. Logistic regression analyses identified factors associated with tobacco use. Overall, 4691 people were included in this analysis.The prevalence of any tobacco use was 19.8% (95% CI: 18.4–21.2). Tobacco use was higher for men (29.2% [27.0–31.5]) than women (11.8% [10.3–13.4]). The prevalence of smoked tobacco was 11.3% (10.3–12.4), with a significantly higher prevalence among men (24.5% [22.1–27.0]) than women (0.1% [0.01–0.3]). The overall prevalence of other tobacco use was 8.9% (7.4–10.7), with lower values for men (5.6% [4.1–7.2]) compared to women (11.7% [9.4–14.1]). Tobacco smoking among men was significantly associated with an increased age and alcohol consumption. The analysis of risk factors for other tobacco use stratified by gender showed that age, education, residence, and alcohol consumption were significantly associated with consumption for women, and age and alcohol consumption for men.Tobacco use is common in Burkina Faso. To effectively reduce tobacco use in Burkina Faso, a comprehensive tobacco control program should consider associated factors, such as gender, age, and alcohol consumption. Despite considerable efforts, tobacco use remains a leading cause of preventable deaths worldwide, according to the World Health Organization (WHO) . TobaccoWhile the prevalence of tobacco use has been low in Africa compared to other regions, future projections show rapid increases in smoking, particularly among men, and an increase in the burden of non-communicable disease in the region, accentuating the need for country-level data on the prevalence of smoking and SLT use . AccordiIn response to concerns about morbidity and mortality associated with tobacco consumption, the WHO created the Framework Convention on Tobacco Control (FCTC) in 2005 and “MPOWER” measures in 2008 as a set of low-cost, high-effective measures to control tobacco use . The useAccording to 2011 Demographic and Health Survey data, the prevalence of smoking among a representative sample of adult men in Burkina Faso was 21.2% and among adult women, was 0.09%, with 3.86% reported SLT use among women. No SLT data was collected from men in this survey . The onlε absolute error (5%); zα fractile of normal distribution of 5% error (1.96)) [sub)  and for a non-response rate (t) of 20%. The Burkina STEPS survey consisted of 4800 people, and 4691 participants were included in our analysis, after excluding observations with missing data about tobacco consumption. The response rate of the STEPS survey was 99.1% in Burkina Faso [We analyzed cross-sectional data from the first Burkina Faso nationwide STEPS survey conducted in 2013 by the Ministry of Health of Burkina Faso with technical support from the WHO. STEPS is a population-based health survey administered to people aged 25–64 years in many countries worldwide that uses stratified three-stage cluster sampling proportional to size to select participants. The sample size is estimated using the following Schwartz formula:  (1.96)) , 12. TheThe study’s sampling frame was based upon enumerations (EAs) from the 2006 general census of the population and housing (GCPH) and updated in 2010 during the Demographic and Health Survey in Burkina Faso . In the The STEPS questionnaire is made up of several modules that include demographic information, anthropometric measures, and behavioral measurement. A full description of the study design and the data collection has been published elsewhere , 14. AllThe protocol of the STEPS survey was reviewed and approved by the Ethics Committee for Health Research of the Ministry of Health, which gave clearance in accordance with regulations in force (Deliberation No. 2012–12- 092 of 05 December 2012). Written informed consent was systematically sought and obtained from all participants before inclusion in the study. The confidentiality of study participants was fully respected and the analyses performed did not identify any participant.Outcomes of interest include three measures of tobacco use: current any tobacco use, current cigarette smoking, and current other tobacco use (SLT). Current cigarette smoking was assessed with the question ‘During the past 30 days, how many days did you smoke cigarettes?’, with current smoking defined as smoking on at least 1 day during the past 30 days. Current SLT use was assessed with the question ‘During the past 30 days, on how many days did you use any smokeless tobacco products?’, with current SLT use defined as use of any SLT product on at least 1 day in the past 30 days. Any tobacco use is defined as either current cigarette smoking or other tobacco use in the past 30 days.Independent variables included demographic characteristics such as age, sex, gender, marital status, place of residence, and education level, as well as behavioral measurements such as alcohol consumption Table .Table 12 test or Fisher exact test, when appropriate. Multivariable logistic regression models were used to estimate the associations between tobacco consumption, either smoking or other tobacco consumption, and other factors. A two-sided p value of less than 0.05 was considered statistically significant. We used QGIS to represent the variation of prevalence between the different regions of Burkina Faso.All data were weighted according to the cluster sampling design of the survey using strata and primary sampling units to allow the sample to be nationally representative. We used the Complex Samples module in Stata version 15 for data analyses. Weighted prevalence values (with corresponding 95% CIs) were calculated by sex and age group. We tested differences in prevalence with the χOf the 4800 people originally sampled for the survey, 84 were excluded because they were not home after two visits, were not willing to participate in the study, or had no information available on tobacco use. The data of 25 people were deleted due to incomplete information about sampling weights in the database. The response rate for this analysis was 97.7%. Table p = 0.001)). Tobacco use was highest in the Centre-Nord region (40.9% (95% CI: 29.3–53.6)) and lowest in the Centre-Est region (7.1% (95% CI: 3.7–13.2)). See Fig. The overall prevalence of any tobacco consumption was 19.8% (95% CI: 17.8–21.9). The prevalence among males and females was statistically different, with a value of 29.2% (95% CI: 26.4–32.1) vs. 11.8% (95% CI: 9.6–14.3), respectively. Tobacco use was also statistically higher in rural areas (21.8% (95% CI: 19.4–24.3)) than in urban areas  of participants reported current smoking. The prevalence of smoking was also highest in the Centre-Nord region (17.3% (95% CI: 13.0–22.7)) and lowest in the Centre-Est region (3.9% (95% CI: 1.7–8.6)). The prevalence of daily smoking was 9.3% (95% CI: 8.3–10.6).In total, 8.9% (95% CI: 7.4–10.7) of participants reported using smokeless tobacco (SLT) products. The Centre-Nord region had the highest prevalence of SLT use (25.2% (95% CI: 16.5–36.5)), and the Cascade region had the lowest prevalence (2.4% (95% CI: 0.7–7.8)). The prevalence of smoking was higher in men than in women (24.5% (95% CI: 22.1–27.0) vs. 0.07% (95% CI: 0.0–0.2), respectively). Women used SLT more frequently than men (11.7% (95% CI: 9.4–14.1) vs. 5.6% (95% CI: 4.1–7.2)). Smoking and SLT use were more frequent in rural areas than urban areas, with values of 11.6% vs. 10.4 and 10.8% vs. 3.7%, respectively. The prevalence of daily SLT use was 7.8% (95% CI: 6.5–9.5).n = 2256). Table p = 0.79), education level (p = 0.22), marital status (p = 0.76), or occupational status (p = 0.07). Risk factors independently associated with tobacco smoking were sex (p < 0.001), age (p = 0.021), and alcohol consumption (p < 0.001). The prevalence of smoking decreases with age , but only two of the 2435 women included in this study reported smoking. Therefore, we limited the analysis of smoking and associated factors to the male population only (p < 0.001) There is also a statistically significant difference between men and women (p < 0.001): the prevalence for women is 11.7% (95% CI: 9.4–14.1) and is 5.6% (95% CI: 4.1–7.2) for men. Among women, risk factors associated with SLT included age (p < 0.001), education level (p < 0.001), living area (p < 0.001), and alcohol consumption (p < 0.001) Among men, SLT use was associated with age (p < 0.001) and alcohol consumption (p < 0.001) . The prevalence is 1.9% (95% CI: 1.2–3.1) in urban residents and 9.7% (95% CI: 8.8–10.7) in rural residents. The difference in SLT prevalence between living areas is statistically significant  and ischemic heart disease. Health effects of SLT vary by region, related to the types of tobacco that are used in those regions . In Sub-Concerning the limitations of this study, we report here the results of the first nationally representative survey on the prevalence and risk factors for tobacco consumption in Burkina Faso. The first limitation stems from the cross-sectional nature of the data, which limits the possibility of deriving causal inferences. The second limitation is that tobacco and alcohol consumption were obtained during interviews and are therefore dependent on the faith of the participants. There is thus both a risk of memory bias and social desirability. It can therefore be estimated that the numbers and prevalence obtained in this survey underestimate the actual consumption. The last point is that some well-known risk factors for tobacco consumption were not included in the study because data on these variables have not been collected during the STEPS survey. One of these variables is the socio-economic status. Given the study design (cluster sampling design) and the sample size, the results of this study can be extended to the whole of Burkina Faso.Based on the results of this study, important preventive measures need to be taken to reduce tobacco consumption in Burkina Faso, with targeted approaches to sections of the population most affected by different types of tobacco use. In general, efforts should target tobacco use in youth, smoking among men, and SLT consumption among women and in rural areas.Two important aspects should be taken into consideration when organizing tobacco prevention campaigns in Burkina Faso. The first is the necessity to integrate tobacco and alcohol campaign programs, since we have seen that both consumptions are highly correlated for both smoked and SLT consumption. The second is to pay more attention to SLT use in tobacco control programs as it constitutes a significant part of the tobacco use in Burkina Faso and is the principal type found in women.The prevalence of tobacco consumption remains high in Burkina Faso, despite restrictive measures and control plans adopted in recent years. Since tobacco consumption is an important risk factor for cardiovascular diseases and other NCDs, it is important to promote and accelerate the implementation of various measures to decrease consumption, especially SLT consumption, in future years. Further spatial analyses could be useful to identify areas of high tobacco consumption in order to focus more on these areas in tobacco control and prevention program planning.


Toxoplasma gondii from camels by bioassay method in mice model and detect parasitic DNA in brain mice by molecular methods.We isolated Toxoplasma antibodies by modified agglutination test (MAT) test. Bioassay method was done in positive MAT blood camels in BALB/c mice and Nested PCR performed in seropositive tissue samples to amplify the B1 and GRA6 genes. The existence of polymorphic restriction sites for endonuclease MseI was used with PCRRFLP method and Sequencing analysis to evaluate the prevalence of type strains One hundred tissue samples including heart (n=50), and diaphragm (n=50) were collected from camels (n=50) slaughtered in abattoirs from Feb to Oct 2015 in three provinces located in eastern Iran. In first, blood sample from 50 camels was assayed for anti-T. gondii B1 gene, including 7(14%) diaphragm, 6(12%) heart. Moreover, 3(6%) tissue samples of camels were found positive with GRA6 gene for T. gondii. There are three genotypes and mix genotype using MseI enzyme among all positive samples.Overall, 13 (26%) of camels were positive with titer of 1:20 for toxoplasmosis and 13(26%) tissue samples of camels were found positive for the T. gondii with previously recognized genotypes in the tissues of camels for first time from Iran. Since consumption of meat camels are raising in Iran, there may be a high risk of toxoplasmosis through consumption of products from these hosts due to their susceptibility to the infection.The obtained results from serological and molecular tests demonstrated the infection of  Toxoplasma gondii, is one of the most common parasitic infections of human and a wide spectrum of warm-blooded animals. It has been found world-wide from Alaska to Australia  and diaphragm (n=50) as well as 50 blood samples were obtained from camels (n=50) slaughtered in abattoirs from three provinces located in eastern Iran including Kerman, Razavi Khorasan, and south Khorasan Provinces, Iran between Feb to Oct 2015. All the collected animals had been born and raised in southeastern Iran and were intended for human consumption.T. gondii infection using the Modified agglutination test  (All blood samples from camels were examined for  France) . Sera weT. gondii  for another 30 sec. After homogenization, 250 ml of a pepsin solution  was added. After an incubation of 1 h at 37 °C, the homogenate was filtered through two layers of gauze and centrifuged at 1200×g for 10 min. The supernatant was discarded and the pellet was resuspended in 15 to 20 ml of 1.2% sodium bicarbonate solution (pH=8.3) and recentrifuged at 1200×g for 10 min. The supernatant was discarded and the sediment was re-suspended in 5 to 10 ml of antibiotic saline solution . One ml of this suspension was inoculated intraperitoneally (IP) in five BALB/c mice per sample (Approximately 50 gr of heart and diaphragm tissue samples from modified agglutination test (MAT) positive camels were processed for isolation of . gondii . Brieflyution 0.1 M NaCl fT. gondii seronegative female BALB/C mice, obtained from the animal facility of the Kerman University of Medical Sciences . Non-infected mice (n=5) were kept separately as negative controls. The mice were given commercial pelleted feed and municipal chlorinated water ad libitum. The inoculated mice were observed daily for the presence of clinical signs until day 60 post inoculation. A T. gondii isolate was considered virulent if mortality of mice was observed within two weeks of infection (T. gondii-antibodies by the modified agglutination test (MAT).Bioassays were performed within 1 to 3 d after the slaughter of the animals. The mice used were nfection . The micT. gondii cysts were detected in any of the five inoculated mice.Two months after intraperitoneal (IP) inoculation, the brain from surviving mice was removed after euthanasia with di-ethyl ether. Each brain was homogenized in 1 ml PBS (pH 7.2) using a mortar and pestle. Following microscopic examination and counting of cysts, the homogenates were stored in 1.5 ml Eppendorf tubes and frozen at −20 °C until DNA extraction was done. The number of cysts in three aliquots of 10 μl each was counted under a light microscope with a 100x magnification. The total number of cysts in the brain of each mouse was determined by converting the sum of cysts in 30 μl to the whole volume of the brain homogenates ,16. A biDNA was extracted from mice brain . Using tThis research project was approved by the Ethics Committee of the Kerman University of Medical Science. All efforts were made to minimize animal suffering during the course of the study (No. 94/389).2 concentration adjusted to 1.5 mM; 100 ng/μl of DNA was used for the PCR assay. Each sample was tested in duplicate for each method. The nucleotide sequences of the primers for the nested-PCR assays targeting the B1 and GRA6 gene are listed in T. gondii DNA. The first round of PCR amplification contained 10 mMTris–HCl, pH 8.3 (at 25 °C), 50 mMKCl, 1.5 mM MgCl2, 5 μM each primer  of extracted DNA. Reactions were started with an initial denaturation at 94 °C for 5 min and then cycled 30 times with denaturation at 94 °C for 20 sec, followed by annealing at 53 °C for 20 sec for B1 gene and finally an extension step at 72 °C for 20 sec followed by a 5 min final extension at 72 °C. A PCR negative-control sample omitted template DNA, replaced by sterile water and a positive control sample that used extracted DNA from T. gondii tachyzoites RH-strain.Nested-PCR was performed in a final volume of 25 μl using the Taq DNA Pol 2.0 × master mix with an MgClh primer , 250 μM μg/ml; Applichem, Biochemica, Germany). The size of PCR target is expected to yield 791 bp for Gra6 positive reactions.Nested PCR was done to amplify the coding region of the GRA6 gene. PCR amplification was performed with 1 μl of DNA template in 50 μl of a reaction mixture containing 10 mMTris-HCl, pH 8.3; 50 mMKCl; 2 mM MgCl2; 200 μM dNTPs; 0.5 μM each of oligonucleotide primers; 1.25 units of Taq DNA polymerase ; and 50 pmol of each primer. All PCRs were performed in a FlexCycler . The first step of amplification was 5 min of denaturation at 94 °C. This step was followed by 35 cycles, with 1 cycle consisting of 30 sec at 94 °C, 60 sec at the annealing temperature for each pair of primers, and 90 sec at 72 °C. The final cycle was followed by an extension step of 7 min at 72 °C. The PCR primer pair was designed from the GRA6 gene sequence , GRA6R: MseI restriction endonuclease ,  for digestion. Briefly, 15 ml of PCR product was digested using 1.5 U of MseIenzyme and 2 U buffer R and incubated at 65 °C for 4 h in accordance with the manufacturer’s protocol. The restriction fragments were separated by electrophoresis in 2% agarose gel followed by staining with ethidium bromide and visualization under UV. The cut position of MseI in GRA6 genes of types I, II, and III was 168 bp and 712 bp, 71 bp and 694 bp, and 71 bp, 168 bp, and 712 bp, respectively . Nested PCR products and internal primers were sent to Macrogene Company of South Korea. Results were aligned with BioEdit and sequence Scanner program and compared to the following sequence data available from GeneBank: AB235428, AB235433, AF239283 RH type I, AF239284, AB235430 Beverley type II and AF239286, AB235429NED type III, (GenBank). The phylogenetic relationships among genotypes were estimated using Maximum-likelihood analysis. Mega 6 software was also used to construct the phylogeny tree to compare our isolates with types submitted in Genebank. Hammondia hammondi (GenBank accession numbers: XM 008888391) was employed as the outgroup to root the resulting trees.Sequence analysis for comparison of B1 and GRA6 genetic profiles were performed to determine type of Among 50 blood sample of camels, 13 (26%) were positive. a titer of 1:20 was considered positive and the tissue samples of positive isolates were collected for bioassay method.T. gondii B1 gene, including 7 (14%) diaphragms, and 6(12%) hearts. Among positive samples, 9(18%) and 4(8%) were male and female respectively.T. gondii, including 2(4%) diaphragms and 1(2%) heart, of which, 2(4%) were male and 1(1%) was female. In the present study, total prevalence with the main marker of diagnosis in tissues, B1 gene, was 26% (Only 3(6%) tissue samples of camels were found positive with GRA6 gene for  was 26% .MseI enzyme among all positive samples (To determine the genetic characterization of isolates, genotyping of positive samples was examined by PCR-RFLP with GRA6 gene. As shown in  samples .The phylogenetic analysis demonstrated that three samples 22, 23 and 24 were identified similar to type 2 with high similarity in sister clade . RepreseT. gondii infects nearly one-third of the world population. Although toxoplasmosis is asymptomatic in immunocompromised individuals, the disease is life-threatening during pregnancy and in immunocompromised drones (d drones .T. gondii mainly via ingesting raw or undercooked meat of infected animals or by ingesting food or water contaminated with oocysts and the parasite can be transmitted to the fetus by the passage of tachyzoites through the placenta (T. gondii in milk (Normally, humans become infected with placenta . Camels  in milk  and tiss in milk  of camel in milk . Camels  in milk .T. gondii infection in camels varies depending on the geographical situation (T. gondii infection from camels in Ethiopia. An overall prevalence of 49.62% by DAT and 40.49% by indirect ELISA test were detected (T. gondii infection among childbearing age women in Kerman city used by ELISA method (T. gondii B1 gene (T. gondii antibodies (T. gondii in 3.12% of milk of camels in Iran (T. gondii among camels in Iran. Therefore, this study for the first time was performed on isolation and molecular detection of T. gondii DNA in camels in Iran. In our study, all 13 seropositive samples with MAT test were also identified positive after bioassay by amplification of B1 gene on mice brain and not seen brain cysts of T. gongii by microscopy examination in inoculated BALB/c mice. The nested PCR method shows the high sensitivity and specificity for the detection of toxoplasmosis; furthermore, B1 gene because of having higher copies in genome of T. gondii and also high specificity and sensitivity is as suitable instrument to identify T. gondii (T. gondii isolates using analysis of the pattern derived from the cleavage of its DNA (T. gondii by B1 and GRA6 genes, respectively. In this study to determine the genotyping of T. gondii isolates, GRA6 products were digested with MseI restriction enzyme, and the type of samples was determined by digestion patterns. There are three genotypes and mix genotype using MseI enzymes among all positive isolates.The prevalence of ituation . In Iranituation  to 90.90ituation . A direcdetected . AnotherA method . Recentl B1 gene .Previoustibodies . Another in Iran . However. gondii . On the  its DNA . Here, wIn Iran, type II and III were found in 4 isolates from sheep according to microsatellite and GRA6 gene sequence analysis . MoreoveT. gondii DNA in the tissues of camels from Iran for the first time. Since camels are the most important food sources in Iran, there may be a high risk of contamination through consumption of products from these hosts due to their susceptibility to the infection. Therefore, control of the infection in food animals is important for consumer protection.The findings of this study demonstrated the presence of


Forthcoming legislative changes will legalize and make cannabis widely available in Canada. We conducted an analysis of Ontario’s birth registry to determine recent trends and correlates of cannabis use in pregnancy.We conducted a population-based retrospective cohort study assembled from the Better Outcomes Registry & Network (BORN) Ontario database, covering live births and stillbirths in Ontario between April 2012 and December 2017. Trends in self-reported cannabis use in pregnancy were analyzed according to maternal age and area-level socio-economic status (SES) using log binomial regression analysis.p-trend, < 0.001), equivalent to a relative increase of 61% . The crude prevalence of cannabis use in pregnancy among women aged 15 to 24 years and in the lowest two area-level income quintiles was 6.7%, compared to 0.3% among women aged 35 years and over in the highest three income quintiles . A majority (52.0%) of cannabis users were aged 15–24 years and 54.7% of users were in the lowest two income quintiles.A total of 10,731 women reported cannabis use in pregnancy. Prevalence increased from 1.2% in 2012 to 1.8% in 2017  contains supplementary material, which is available to authorized users. In 2017, the Government of Canada introduced legislation to allow the consumption, sale, and distribution of cannabis (marijuana) for non-medical uses by October 2018  is a comprehensive birth registry in the province of Ontario and encompasses nearly 40% of all births in Canada  version 6, developed by Statistics Canada , yielding a final sample size of 732,818 for the analysis. Excluded participants were older, more likely to report alcohol use in pregnancy, to have diabetes, and higher income  records were excluded due to missing information on cannabis use, maternal age, or area-level income. Records with maternal age less than 15 or greater than 50 were excluded  were aged 25 through 34 years, 13.4% were less than 25 years, and 22.5% were greater than 35 years of age. A majority (69.7%) were from urban areas with greater than 100,000 inhabitants and 24.8%  fell into the highest income quintile based on residential postal code. 1.5%  of women used cannabis in pregnancy and 9.1% used cigarettes at the first prenatal visit (Table p = 0.90). Cannabis use was substantially higher among women who reported alcohol and tobacco use in pregnancy .Among women with singleton live births in the analysis, 64.1% . This equates to a relative increase of 61% between 2012 and 2017  adjusting for maternal age, income, and population size (Table p < 0.001). There was an 18% relative decrease in prevalence with each additional year of maternal age . Cannabis use varied by income, with women in the lowest two quintiles reporting the highest usage  as compared to women in the highest three quintiles  . The relative risk for cannabis use in the lowest compared to the highest income quintile was 5.30 (95% CI 4.95 to 5.67), and attenuated to 3.23 (95% CI 3.02 to 3.46) after adjusting for maternal age, year, and population size.In Ontario, the overall prevalence of cannabis use in pregnancy increased from 1.2% in 2012 to 1.8% in 2017  and upper three quintiles  of area-level income (Table p < 0.001).Among those who reported cannabis use in pregnancy, the majority (52.0%) were aged 15–25 years, although due to increases in overall usage, this proportion declined with time from 60.1% in 2012 to 42.7% in 2017 Fig. . There wThere has been an increase in cannabis use among pregnant women in Ontario since 2012, and the increase occurred predominately among younger women and those of lower socio-economic status (SES). In these groups, the prevalence of self-reported cannabis use during pregnancy was 5.5% and 3.1%, respectively, compared with 1.8% in the overall obstetrical population. Young women under 25 years of age and women from lower area-level SES represented more than half of all women who reported using cannabis during pregnancy, suggesting that the burden of potential health implications of cannabis use in pregnancy may be greater among these women.Previous studies have indicated an association between prenatal cannabis use and adverse pregnancy outcomes such as preterm birth, small for gestational age, and admission to neonatal intensive care ESM 2(DOCX 110 kb)