Simple bone cysts (SBCs) and intraosseous lipoma (IOL) of the calcaneus are rare tumor entities that are primarily diagnosed due to unspecific heel pain, incidental findings, or rarely due to pathological fractures. Compared to traditional open tumor resections, endoscopic resection of these benign tumors aims to minimize surgical morbidity and maximize surgical efficiency without compromising safety. Grafting is regularly performed to reduce the risk of recurrence and stimulate osseous consolidation of the lytic lesion. As the incidence is low and treatment strategies are heterogeneous, there is no clear consensus for the treatment of simple cysts or intraosseous lipomas of the calcaneus. The objectives of this study are (a) to present medium to long-term results after endoscopic resection and grafting with allogenic cancellous bone or bioresorbable hydroxyapatite and calcium sulfate cement, and (b) to add further evidence to the discussion of whether calcaneal SBC and IOL are the same entity at different developmental stages. Between 2012 and 2019, a total of 25 benign bone tumors consisting of 17 SBCs and 8 IOLs were treated by A.T. with endoscopic resection and grafting, comprising the largest cohort to date. For grafting, 12 patients received allogenic cancellous bone (group A) and 13 patients received injectable bone substitute (group B). Pre- and postoperative imaging using plain X-rays and MRI was retrospectively analyzed with a mean follow-up time of 24.5 months to assess tumor size, osseous consolidation (modified Neer classification), and tumor recurrence. A retrospective chart analysis focusing on adverse intra- and perioperative events and other complications associated with the surgical procedure was performed using the modified Clavien–Dindo classification (CD1-3). A total of 12/13 cases with allogenic bone grafting showed a Neer Type 1 osseous healing of the tumorous lesion after endoscopic resection, whereas only 5/11 cases with injectable bone substitute showed sufficient healing (types 1 and 2). There were three recurrent cysts (Neer 4) and two persistent cysts (Neer 3) after using injectable bone substitute. Two CD1 complications were observed in group A  and eight complications were observed in group B . At least two IOLs diagnosed preoperatively using MRI were ultimately identified as SBCs upon histopathologic examination. Allogenic cancellous bone grafting after endoscopic resection of calcaneal SBC or IOL showed a very low rate of complications and no tumor recurrence in our series. On the other hand, depending on the material used, injectable bone substitute showed a high rate of “white-out” (excessive drainage), resulting in multiple complications such as prolonged wound healing, insufficient permanent defect filling, recurrence, and revision surgery. Over time, calcaneal SBC may transform into IOL, exhibiting distinct features of both entities simultaneously during ossoscopy and histopathological analysis. According to WHO’s classification of soft tissue and bone tumors, a simple bone cyst (SBC) is an intramedullary, usually unilocular, cystic bone tumor lined by a fibrous membrane and filled with serous or serosanguineous fluid. The older term, unicameral bone cyst, is no longer recommended . The occcalcaneal simple bone cysts are observed later, usually during the second and third decades of life [calcaneal SBC (n = 42) in our series of foot and ankle tumors was 18.1 ± 6.8. There was a male predominance, with a m:f ratio of 2.2:1 [SBC of the long bones most commonly occurs in children and adolescents, with a reported average age at diagnosis of 9–11 years ,8. On th of life ,11. In a of life . The aveLocalized in the lower extremity, SBCs may cause persistent pain and thus warrant surgical therapy. However, they are diagnosed as an incidental finding in approximately 30% of cases ,14,15. ILipoma of bone is a benign neoplasm composed of white adipocytes arising within or on the surface of bone. Intraosseous lipoma (IOL) commonly arises in the calcaneus and metaphysis of long tubular bones, especially the femur, tibia, and humerus. Approximately 70% of IOL cases involve the lower limb . IntraosIOL can be misinterpreted as plantar fasciitis and other common causes of heel pain ,21,22. ISBC and IOL are similar in many ways, including their location and radiological appearance, but their content differs. SBCs contain fluid, whereas lipomas contain fat, although some lesions may exhibit a mixed content . Both capseudocyst” for this area of decreased radiopacity was first used by Irry et al. to describe these findings [pseudocyst” may be confusing in the setting of simple bone cyst as pseudocyst is not lined by fibrous membrane and is not a true SBC. The pathogenesis of the two entities, SBC and IOL, is a subject of ongoing controversy. In their 2017 publication, Malghem et al. discussed in detail the most common hypotheses regarding the pathogenesis of calcaneal SBC and IOL [The anterior calcaneal location of SBC and IOL corresponds exactly to a triangular anatomic area in which the trabecular network is naturally much sparser  IOL may develop from SBC.n = 17) had a mean age of 17.0y (range 12–41), with 12 male and 6 female patients being affected. Patients with IOLs (n = 8) had a mean age of 38.0 years old at the time of surgery (range 19–62), and there were 6 male and 2 female patients in this subgroup  tumorous lesions which are commonly seen in Diard´s Area 6 and must be distinguished from calcaneal SBC and IOL . Overallsubgroup .Thirteen out of the 25 cases were incidental findings (52%). In 7 of the 13 cases, an MRI was performed after an ankle sprain. The remaining 6 MRIs were performed for the following reasons: achillodynia (2×), after a medial malleolar fracture, insect bite, tarsal coalition, and after whole-body computed tomography in a polytrauma patient . For alln = 10), tumor size with increased risk of a pathological fracture (n = 10), or at the explicit request of the patient in the case of tumor anxiety (n = 5). In cases of heel pain, all relevant differential diagnoses had been excluded through repeated clinical assessment and imaging diagnostics. The critical size for an elevated risk of fracture was defined as 100% of the intracalcaneal cross-section in the coronary plane and at least 30% in the sagittal plane of the lesion, according to the definition of Pogoda [Surgery was indicated for the following reasons: pain  could decide whether to use allogenic bone or injectable bone substitute. Our hypothesis was that the use of injectable bone substitute might decrease the surgery duration and allow for early full weight-bearing. Patients who requested early full weight-bearing were recommended to have IBS as the filling material. Moreover, some patients preferred to have artificial bone substitute over allogenic bone for personal reasons and therefore opted for IBS as a filling material. ACB was recommended for all other patients based on our previous experience regarding osseous integration and the current state of the literature on calcaneal SBC, which suggests that cancellous bone is beneficial in terms of recurrence .Autologous bone grafting was also discussed but not recommended due to the associated donor site morbidity. No randomization was performed.A total of 12 patients with a mean age of 17.9 years (range 12–34) received allogenic cancellous bone , and 13 patients with a mean age of 30.4 years (range 12–62) received injectable bone substitute . In ® Cancellous Chips, LifeNet Health, Vienne, Austria), and injectable demineralized bone matrix was used in 2 cases . Four different types of injectable bone substitute were used: Cerament©  in 10 cases, Pro-Dense©  in 1 case, QuickSet© calcium phosphate bone cement, Arthrex, Naples, FL, USA) in 1 case, and Innotere©  in 1 case.For grafting, allogenic bone in the form of cancellous bone was used for plombage of the cavity in 10 cases  was used for the last case upon the patient’s request.Depending on the product used, curing of the bone substitute differs slightly:For Cerament©, the setting time is specified by the manufacturer as 15 min, and the initial compressive strength is 10–75 MPa (wet conditions–dry conditions). For Innotere©, a setting time of 24 h is given until the compression strength of cancellous bone is reached (10 MPa). For ProDense, the manufacturer specifies a compressive strength of 40 MPa after 2 h in wet conditions. Quickset is reported to reach a compression strength of 24 MPA at 24 h after implantation. After using injectable bone substitute, limited weight-bearing (15 kg) was recommended until wound healing was completed (2 weeks). When using allogenic bone for grafting, 6 weeks of limited weight bearing was indicated.A radiologic follow-up was routinely performed 6 weeks, 12 weeks, and 12 months postop with plain radiography. To rule out recurrence, MRI imaging was indicated for all cases demonstrating a healing result with a defect on conventional radiographs (types Neer B–D).The classification of the osseous healing outcome was based on the modified Neer classification . The modPre- and postoperative imaging with plain X-rays and MRI was retrospectively analyzed to assess tumor size, osseous consolidation, and tumor recurrence. A retrospective chart analysis focusing on adverse intra- and perioperative events and other complications associated to the surgical procedure was performed using the modified Clavien–Dindo classification ,55,56. CThe surgery duration was recorded in minutes. All but one ossoscopy was performed as a standalone procedure. In one case, endoscopic tumor resection was performed in combination with a joint preserving surgery for tarsal coalition. For this case, no isolated surgical time was available for the endoscopic procedure.The surgical technique has been previously described by the authors ,24,58. THartmann ear speculum  was introduced through one of the ossoscopy portals  and range (min./max.). A Fisher´s exact test was used to determine the statistical significance of the differences between Group A and Group B, as well as between the different Neer types of osseous healing. A The mean follow-up (f/u) of 24/25 patients was 24.5 months (range 12–91). One patient (case 21) was lost to f/u after the first control six weeks postop. Patients showing complete osseous healing without any defect (Neer type A) in conventional X-rays and no symptoms one year postop were not required to undergo further imaging controls by our clinic, but were contacted by phone and/or mail in January 2021 asking for any additional imaging that was performed in the meantime. A return postage-paid envelope was mailed to the patients requesting the additional imaging. In two cases, the imaging was transmitted digitally. As a result, five more MRI follow-ups of were acquired for the initial Neer type A cases. Only one case (case 6) with MRI performed 91 months after index surgery showed small areas of recurrent SBC a,b, corrMoreover, where available, the amount of filling material used is noted . For injp = 0.011).Statistically significant differences were observed in the healing outcome between type Neer A and higher Neer types  in favor of the use of allogeneic cancellous bone versus injectable bone substitute , both requiring revision surgery. The first case was caused by iatrogenic damage of the medial calcaneal cortex during endoscopic tumor resection and undetected leakage of the injectable bone substitute (case 6). Due to inadequate pain and swelling on the medial aspect of the hindfoot, CT imaging was performed and revision surgery was necessary to remove the leaked material on the medial side two days after the index surgery. The second Stage 3 complication required open surgical revision surgery (performed elsewhere) for suspected deep infection and severe inflammation of the surrounding soft tissue case 24, . Microbip = 0.0012).In group A .The contingency table was used to show the absolute frequency of use of ACB and IBS, as well as complications between the two groups . Fisher’p < 0.05). For group B, the surgery duration for the isolated ossoscopy was not available retrospectively in two instances (cases 16 and 21). One of those procedures (case 21) was performed in combination with a joint-preserving PCFD correction.Overall, the mean operation time was 75 min (range 3–133) and 72 min (range 35–103) for group A (ACB), and 84 min (range 45–133) for group B (IBS), showing no statistically significant difference  with characteristic preoperative MRI findings were histopathologically diagnosed as SBC. One additional case of MRI-diagnosed SBC showed membranous material with a histiocyte-resorptive reaction, consistent with SBC (case 11). Another case had material from a hemorrhaged lesion along with several bony particles (case 8), but no clear signs of lipoma of the bone. The absence of lipomatous tissue can be explained by the surgical technique. During endoscopic resection of IOL, the lipomatous tissue from the bone cavity is often washed out until a clear ossoscopic view is established and appropriate specimens are obtained see . This prAre simple bone cyst and intraosseous lipoma two different entities?Preoperative imaging with plain radiographs and MRI led to the diagnosis of 17 SBCs and eight IOLs. Both entities exhibited a characteristic, almost pathognomonic appearance on MRI, making bioptic verification of the diagnosis unnecessary. Often times, MRI revealed a mixed form of SBC and IOL, showing lipomatous tissue enclosing central cystic areas . This haWhile MRI allows differentiation between cystic from lipomatous areas of the lesion, histopathological analysis is required to verify the correct diagnosis. Direct endoscopic visualization of the lesion can help to correctly assess mixed and transitional forms of SBC and IOL macroscopically . The higTwo cases initially diagnosed as IOLs based on MRI turned out to be SBC in the histopathological analysis (case 5 and 12). Another case of suspected IOL demonstrated membranous material with a histiocyte-resorptive reaction but no cholesterol clefts  upon histopathological evaluation, resembling SBC (case 10).Both our endoscopic and histopathological results support the theory proposed by other authors such as Malghem, Tins, and Kawaguchi that  IOL develops form SBC ,30,50,64Which option should be used now: allogenic cancellous bone or injectable bone substitute?A systematic review on the treatment of unicameral bone cysts of the calcaneus conducted by Levy et al. from 2015 concluded that open curettage with autograft bone augmentation is the most effective procedure. Unsurprisingly, autograft procedures resulted in significantly greater radiographic healing compared with allografting. The review included only one publication on endoscopic curettage and calcium–phosphate injection, and two more reports with two cases each treated with open curettage and calcium phosphate or calcium sulfate, respectively .45S5 Bioglass was reported to better facilitate the formation of new bone with a faster drying time of the skin incision than allogenic bone in open curettage of 31 patients, including 18 cases of SBC and six cases of IOL with a f/u of 12 months [In a recent study by Ma et al., 2 months . Only plKarr et al. reported the use of Cerament© calcium sulfate/calcium phosphate bone void filler in a patient with bilateral calcaneal SBC, mixing the injectable bone substitute with ACB on one side, demonstrating complete trabecular regeneration at 2.3 years (right foot) and three years (left foot) postoperatively .Aycan et al. reported on intralesional curettage and defect filling with cortico-spongeous allograft in 14 patients with calcaneal intraosseous lipoma. After a mean f/u of 84 months (range 18–108), no recurrence was observed . Ulucay Previously, we reported our findings of endoscopic resection and allografting for four cases of calcaneal IOL and six cases of SBC, all demonstrating complete osseous healing after a mean f/u of 19.8 months (range 9.5–39.3) .In 2011, Yildirim performed a comparative study including 26 cases of calcaneal SBC. Thirteen feet underwent open surgical curettage, and 13 cases underwent endoscopic curettage. Both groups received allografting. The operating time and mean length of stay were significantly shorter in the endoscopic group. The time to healing was similar in both groups. The overall success rate was higher for the endoscopic group , and there were no statistically significant differences regarding radiological healing .Similarly, Nishimaura et al. published their findings on 16 calcaneal SBCs in 2016. Eight patients underwent an open procedure, and eight cases were treated with an endoscopic procedure. No significant difference between the two groups was observed in the operative time. The surgery duration for the endoscopic group was 56.1 ± 13.8 min, and no adverse effects were observed. However, the open group experienced one temporary irritation in the sural nerve area and one calcium phosphate cement leakage along the peroneal tendon sheath. The interval for a return to sports was significantly shorter in the endoscopic group. The authors concluded that endoscopic surgery is a useful approach for the treatment of calcaneal bone cysts, allowing early rehabilitation and an early return to sports without any adverse effects.For better comparison, A publication by Choi et al. on endoscopic tumor treatment of the skeleton was not included because the author failed to indicate the anatomic location of nine cases of SBC . SurprisApart from the publication of Nishimura et al., only Farouk et al. provided information on their surgery duration for three cases of endoscopic resection of calcaneal IOL . Grafting was performed in 2/3 cases with PMMA, while the third case did not receive any filling of the bone cavity. The mean surgery time in our series was 75.2 min. (range 35–133) and 71.7 min (range 35–103) for group A (ACB), and 84.2 min (range 45–133) for group B (IBS). Our hypothesis that the use of injectable bone substitute would shorten the duration of the procedure compared with allogeneic bone grafting was not confirmed.Complications were predominantly related to the use of Cerament©, leading to a 90% complication rate (9/10) in patients treated with this particular injectable bone substitute. These complications included DWH with “white-out” in 7/10 cases, one case of DWH without “white-out”, one revision surgery, and one suralis neuropraxia. As the “white-out” phenomenon has been repeatedly observed in various non-tumorous indications in our clinical practice, and excessive white drainage after the use of Cerament© does not seem to be limited to its use in aneurysmal bone cysts, we have stopped using this product entirely. n = 6) or flat bones (n = 1), and not in the calcaneus [Aiba et al. proposed the use of curettage without additional grafting for simple bone cysts in various locations. They reported a recurrence rate of 18.9% (7/37 patients) when using endoscopic curettage as a stand-alone procedure. It is important to note that all recurrences occurred in tubular  ,58.Although this is the largest case series on endoscopic resection of calcaneal SBC and IOL, identifying an exact difference between the two groups  would require much larger sample sizes. This is true for most rare diseases and is unlikely to change in the foreseeable future. Calcaneal SBC proved to be the most common benign bone tumor in our publication on the distribution patterns of foot and ankle tumors  , but it One limitation is the use of different types of injectable bone graft substitutes, preventing any general recommendation for or against their use in calcaneal ossoscopy. Nevertheless, IBS regularly showed prolonged wound drainage (Cerament©) or showed no signs of osseous integration after several years of f/u . The follow-up was inconsistent, both in terms of the type of imaging and the duration of follow-up. MRI was only routinely indicated if plain radiographs at 12 months post-op suggested incomplete healing. Still, 10/25 patients (40%) had (repeated) MRI with a mean f/u of 40.3 months (range 12–91) after surgery, providing a medium-term radiological outcome including MRI for a rare condition treated with an even rarer surgical procedure.Most publications on calcaneal IOL consist of case reports with one or two cases. To the best of our knowledge, there are only three publications with a larger case load of calcaneal IOL, all of them reporting open surgical tumor resection ,67,83. RAnother limitation of this study is its retrospective, non-randomized design. Conducting a prospective, single-center study with sufficient statistical power is not feasible within a reasonable time frame for rare diseases such as calcaneal IOL or SBC. Patients were able to choose the respective filling material after careful consideration of possible advantages and disadvantages. Although both groups (ACB vs. IBS) had almost identical sample size, the inclusion of two different tumor entities could be criticized. Several publications and our own findings suggest that SBC undergoes a continuous lipomatous evolution over time, eventually transforming into IOL ,64,84,85We conclude that endoscopic treatment of calcaneal SBC and IOL is a safe procedure with low complication rates when combined with the correct filling material. The use of Cerament© for calcaneal SBC and IOL is discouraged due to the high complication rates, particulatly the occurrence of “white-out” and cyst recurrence. Other injectable bone substitutes demonstrated no prolonged wound drainage but also no signs of osseous remodeling on repeated MRI examinations over the course of several years.Over time, calcaneal SBC may transform into IOL while exhibiting specific radiologic, endoscopic, and histopathologic features of both entities next to each other.


Mycobacterium avium subsp. paratuberculosis is also used as a tool in some paratuberculosis control programs. The effects of the whole-cell heat-killed Silirum® vaccine on bacterial shedding in the feces and serological response were studied in a controlled field study involving seven French dairy herds, where calves were vaccinated at various ages. A statistically significant reduction of the probability of fecal shedding was observed for cows vaccinated before 4 to 5 months of age compared to non-vaccinated controls. A strong effect of age at vaccination on the serological status investigated in adulthood was also demonstrated, which was also associated with a difference in the protective effect of vaccination.Paratuberculosis is an economically important disease in ruminants, and control in affected herds/flocks primarily relies on test and cull strategies and good hygiene and management practices. Vaccination against the causative agent Mycobacterium avium subsp. paratuberculosis (Map), it is considered an effective tool for paratuberculosis in infected herds. The objectives of this controlled field study were to evaluate the effects of the whole-cell heat-killed Silirum® vaccine on Map fecal shedding and serological status in dairy herds infected with paratuberculosis. (2) Methods: The serological status (ELISA) and fecal shedding (qPCR) of 358 vaccinated cows were assessed over 3 years in 7 infected dairy herds in the Meuse department, France. Within each herd, cows from the last non-vaccinated birth cohort (n = 265) were used as controls. The probability and level of Map fecal shedding and the serological status were modeled using multivariable mixed general linear regression models. (3) Results: Overall, 34.7% of cows tested positive at least once on fecal qPCR, with significant differences between herds, but high shedding levels were observed in only 5.5% of cows. Compared to non-vaccinated seronegative cows, a statistically significant reduction in the probability of Map shedding was found only in cows vaccinated before 4 to 5 months of age that tested negative for Map antibodies throughout the study period , but no significant effect of vaccination on the amount of Map shedding could be evidenced. Finally, the younger the cows were when vaccinated, the less they tested positive on the serum ELISA. (4) Conclusions: a beneficial effect of vaccination on Map fecal shedding may exist in cows vaccinated before 4 to 5 months of age. The variability of the serum ELISA response in vaccinated cows remains to be investigated.(1) Background: paratuberculosis is an important disease in ruminants, causing worldwide economic losses to the livestock industry. Although vaccination is known not to prevent transmission of the causative agent  Mycobacterium avium subsp. paratuberculosis (Map) [Paratuberculosis (PTB), also known as Johne’s disease, is a severe slow-developing, and incurable proliferative enteritis of cattle, sheep, goats, and other domestic and wild ruminants caused by the intracellular bacterium is (Map) . Animalsis (Map) , a longeis (Map) , and an is (Map) ,5. The cis (Map) , which ris (Map) ,8,9. Numis (Map) . At the is (Map) ,12,13. His (Map) . Moreoveis (Map) . Amongstis (Map) . Althougis (Map) ,18,19,20® vaccine was discontinued in 2001 [® vaccine  was approved in 2014. Its use is, however, only allowed in herds deemed to be free of bovine tuberculosis and where the presence of Map has been confirmed and is subject to authorization by the veterinary authorities. Silirum® is a whole-cell heat-killed Map (strain 316F) in an oil adjuvant. Field studies reporting its effect on the epidemiological dynamic of PTB are scarce and almost all come from the Basque Country, Spain, where it has been widely applied for almost two decades. A significant reduction in the number of culled cows showing lesions associated with PTB and with diffuse or severe PTB lesions was found in vaccinated cows compared to non-vaccinated controls in a single herd where cattle were vaccinated regardless of their age or clinical status [® vaccine , related to the Silirum® vaccine and approved for use in sheep and goats, has been intensively used in Australia for almost two decades, and is effective in reducing the incidence of clinical cases, the prevalence of lesion attributable to ovine paratuberculosis, and the prevalence of Map shedding in affected flocks [Vaccination against paratuberculosis was first described as early as 1926 , at whic in 2001 , vaccinal status . Similarl status . Juste el status  reportedp strain 6F in an l status . The Gudd flocks ,34.®-based vaccination programs on Map fecal shedding and serological response in a subset of these herds. By comparing a cohort of vaccinated and unvaccinated (control) cows reared under the same management conditions within each herd, we sought to evaluate vaccination as a management tool in paratuberculosis control programs.At the initiative of the Groupement de Défense Sanitaire de la Meuse (GDS Meuse), a voluntary vaccination program was launched in 2015 in the Meuse administrative department, France, as a potential aid to control losses associated with paratuberculosis in affected herds already engaged in a control plan. The aim of the present study was, therefore, to evaluate the effects of Silirum® was approved in France in 2014. The paratuberculosis control plan relies on hygiene and management measures aiming at reducing calf exposure to Map, including calving in a dedicated paddock, the immediate removal of calves from their dam after birth (before suckling), feeding with colostrum from a dam that tested negative for Map antibodies, and subsequently, with milk replacer or pasteurized waste milk, raising in separate housing with dedicated equipment, and, after weaning, grazing on dedicated pastures with no contact with adult cattle or adult manure. Before vaccination, test-and-cull measures based on the serological testing of all cows over 24 months of age every six months were also implemented. After the introduction of vaccination within each herd, test-and-cull measures were maintained only for non-vaccinated cows. On some occasions, fecal qPCR was performed to evaluate the shedding status of non-vaccinated cows that tested positive for Map antibodies or on vaccinated cows with clinical suspicion of paratuberculosis as a guide for removal from the herd. Seven dairy herds were selected on a voluntary basis. Herds were infected with Map and involved in the paratuberculosis control plan managed by GDS de la Meuse for between 6 to 18 years at the beginning of the current study. They were amongst the first to join the paratuberculosis vaccination plan launched by GDS de la Meuse after SilirumAll herds were free from tuberculosis, Prim’Holstein herds with free-stall housing for lactating cows, ranging in average size from 81 to 302 cattle over 24 months old and for which vaccination against paratuberculosis had been authorized by the French veterinary services.® vaccine bottle ). This vaccination schedule explains the variation in age at vaccination within each herd. Each calf received a single dose of 1 mL of Silirum®, administrated subcutaneously by an accredited veterinarian. Animal id and vaccination date were recorded on a database managed by the GDS de la Meuse.In the selected herds, vaccination against paratuberculosis was initiated between April 2015 and April 2016, and was performed only in young animals. For logistic and convenience reasons, at the first vaccination date within each herd, all calves older than 1 month and younger than 12 months were vaccinated. Subsequently, all calves older than 1 month were vaccinated when their number reached a multiple of 5 . Because the current study was initiated in April 2018, 2–3 years after the start of vaccination depending on the farm, some cows meeting these criteria had not been vaccinated (last birth cohort of unvaccinated cows) and were included as intra-herd controls. Overall, in this setting, 358 vaccinated and 265 non-vaccinated cows were included. A one-sided power calculation, assuming that 25% of unvaccinated cows would shed Map in their feces, indicated that these numbers allowed for a one-third reduction in shedding frequency in vaccinated cows to be evidenced, with a statistical power 1 − β = 80% and a type I error α = 5%.All farmers participated voluntarily and gave written consent that the test results would not be shared with them until the end of the study to avoid bias due to cows being removed from the herd based on a positive result. Information related to cows  was extracted from the French Base de Données National d’Identification (BDNI).® System). Feces and serum samples were frozen at −20 °C before analysis, which was carried out within two months from sampling. Farms were visited on 5 occasions between May 2018 and January 2021 at 6-month intervals, except for the last sampling date, which was delayed due to the COVID-19 lockdown in France. However, because not all cows had entered their second lactation by the first two sampling dates , and considering the high renewal rate in dairy herds, the total number of samples collected per cow was typically between 1 and 3 see . A handfBlood and feces samples were collected by accredited veterinarians as part of the routine paratuberculosis control program managed by GDS de La Meuse. All animal owners gave written consent for samples collected from their animals as part of the paratuberculosis control plan managed by GDS de la Meuse to be used in this study. In this context, ethical approval for animal procedures was deemed not necessary by the Ecole Nationale Vétérinaire de Toulouse ethical committee.®, IDVet, Montpellier, France) was applied to serum samples using an overnight incubation protocol following the manufacturer’s instructions. All samples were tested in duplicate and the average sample-to-positive (S/P) ratios were used as the final result. Duplicates showing discordant results were systematically retested. Negative and positive controls provided by the manufacturers were included on each ELISA plate, and the manufacturer’s guidelines were strictly followed for the interpretation of S/P ratio results: serum samples with S/P values < 60%, between 60 and 70%, and ≥70% were considered negative, doubtful, and positive for Map antibodies, respectively. A commercial ELISA test  for 30 s at 6800 rpm  three times in a bead beater . Magnetic bead-based DNA extraction was performed on a Kingfisher Flex® magnetic particle processor , following the NucleoMag 96 tissue protocol , with the addition of an extraction control  in each plate well. Samples were subjected to qPCR , following the manufacturer’s instructions. Each sample was also tested for amplification of the internal control. Bi-distilled water and synthetic IS900 DNA provided in the amplification kit were used as negative and positive controls, respectively. In addition, fecal samples from a cow at the clinical stage of paratuberculosis with extensive multibacillary intestinal lesions and from a clinically healthy vaccinated but persistently shedding ewe with only focal paucibacillary intestinal lesions were systematically included in each test plate, following an identical preparation process to that of tested samples. Fifty amplification cycles were performed on a LightCycler 96 , and fluorescent signals were recorded in two channels, with FAM detecting IS900 and VIC detecting the extraction control. Due to the overlapping spectra of the two dyes, a color compensation step was applied. Raw fluorescence data were obtained from the LightCycler 96 and modeled using the qpcR package [First, fecal samples underwent a concentration procedure using the ADIAFILTER system  following the manufacturer’s instructions. Ten grams of feces was rehydrated for 1 h in 70 mL of bi-distilled sterile water. The top 10 mL of the supernatant was then filtered and centrifuged using the ADIAFILTER package  in R sof package . Cycle tAll DNA amplifications were ran in duplicate in separate plates, and the average cycle count  was used as the final result. Duplicates yielding discordant results  were further retested. Samples that reached fluorescence with a Ct value below 40 were considered positive. A careful examination of late fluorescence curves indicated that they were associated with low but unambiguously positive results up to 40 Ct, while non-specific amplification results could not be ruled out beyond this threshold. 8 to 10 copies of the IS900 gene per qPCR plate well, using the DNA amplification protocol, as described above. This quantification curve was used to approximate the amount of Map in each qPCR-positive fecal sample.An external quantification curve was constructed using triplicate end-point dilutions of a purified Map culture of the reference K10 strain ATCC 19698 from 10t-test or the Wilcoxon–Mann–Whitney test for ranks when the distributions were strongly skewed or when the sample size was small. A Benjamini–Hochberg correction for multiple comparisons was applied when needed. Frequencies and detection rates were compared using the Fisher exact test. Quantitative variables were compared using the Student −1). Because the distribution of the response variable was strongly skewed to the right, a gamma error distribution with a log link was used. Fixed effects included age at vaccination (AGEVACC), age at sampling (AGESAMP), days in milk within the sampling lactation (DIM), and the serological status at the time of sampling (SEROSTAT). The effect of quantitative variables  was modeled using natural B-splines to ensure smoothness in the relationship between the quantitative variables and the outcome or by creating dummy variables with different cutoff values when appropriate. To account for the clustered structure of the data , cow and farm random effects were included. Logistic mixed-effect models were fitted to data available from all cows to model the effect of vaccination on the probability of Map shedding, indicated by a positive fecal qPCR result. The subset of qPCR-positive fecal samples was used to model the effect of vaccination on Map shedding levels, expressed as log10 estimated number of Map per gram of feces  . Generaln = 365, 58.6%) were sampled twice, and the proportion of cows sampled only once was significantly higher (p = 0.018) amongst the vaccinated cows (31.3%) than the non-vaccinated ones (22.7%)  . p < 10−6). More details on age at sampling and time since vaccination are provided in The median age at first sampling was 48.0 months (first quartile Q1 = 43.8 months and third quartile Q3 = 53.0 months), and, overall, 254 out of the 1163 samples (21.8%) occurred in cows older than 5 years. On average, vaccinated cows were 7.5 months younger that non-vaccinated controls at the different sampling dates . Significant differences were evidenced across herds (p < 2 × 10−5), with cows in Herd A vaccinated at a median age of 6.1 months , and those in Herd D at a median age of 3.1 months .The selected cows had received a single dose of Silirumn = 151) of serum samples were positive for Map antibodies, and 1.7% (n = 20) were doubtful. The proportion of positive or doubtful results was significantly higher among vaccinated animals (23.7%) than among non-vaccinated animals (3.8%) (p < 10−6).Overall, 13.0% (p = 0.001). This decrease was observed in 39 out of the 60 cows, while for 7 other cows an increase in the S/P values was observed, which was greater than 50%. Conversely, among the 186 cows tested at least twice and that yielded a negative ELISA result on their first sampling date, a slight but significant increase in the S/P value was observed, on average, between their first and last test . An increase in S/P values was observed in almost half of these cows , and was greater than 50% in 60 of them, while a decrease higher than 50% occurred in 55 other cows. In the 246 vaccinated cows sampled at least twice, 167 (67.9%) remained negative for Map antibodies, 35 (14.22%) were always positive, 25 (10.2%) changed from positive to negative, and 19 (7.7%) inversely changed from negative to positive. For these 19 cows, seroconversion was detected 38.6 +/− 4.3 months after vaccination . On average, in the 60 cows tested at least twice and that yielded a positive ELISA result on their first sampling date, a significant decline of the S/P value was observed between their first and last test .In the 205 non-vaccinated control cows sampled at least twice, only 10 (4.9%) cows seroconverted and 1 (0.5%) changed from positive to negative; all other cows remained negative on all sampling occasions. On average, the variation in the S/P values between the first and last serological test was significantly positive .The proportion of initially negative cows that mounted an antibody response towards Map that could be detected by ELISA did not significantly differ between vaccinated and non-vaccinated cows (n = 20) precluded any formal statistical comparisons between herds. Seropositive cows were observed in all herds. The proportion of vaccinated cows that tested positive or doubtful for Map antibodies at least once differed significantly between herds, from 13.3% (Herd D) to 68.7% (Herd G). These proportions ranged from 0% (Herd C) to 14.3% (Herd E) for non-vaccinated cows, but the overall small number of ELISA-positive cows (The interplate coefficient of variation (cv) of Ct values for the positive control of the qPCR kit and the external positive fecal controls  were 3.2%, 2.7%, and 3.1%, respectively.−1 of feces) (−1 of feces), and 26 had an estimated Map concentration lower than the clinically healthy shedding ewe control sample . Overall, 216 out of 623 cows (34.7%) (38.3% of vaccinated cows (n = 137) and 29.8% of non-vaccinated ones (n = 79), p = 0.03) tested positive at least once during the study period, but this overall result does not account for the unbalanced distribution of cows between herds. Of the 1160 fecal samples tested, 276 (23.8%) were positive by fecal qPCR. However, only 36 of them (12.9%), originating from 34 cows, were considered highly positive  . Six samOut of the 246 vaccinated cows that were sampled at least twice, 146 (59.3%) consistently tested negative for fecal qPCR and 33 (13.4%) consistently tested positive, while the remaining 82 cows (33.3%) had varying results across the different sampling points, either testing positive or negative. In the 205 non-vaccinated control cows sampled at least twice, 141 (68.8%) were consistently negative and 21 (10.2%) were consistently positive.−1 of feces) were found in all herds except Herd C. The detailed distributions of fecal samples and cows according to herd, vaccination status, and estimated Map concentration are provided in Cows that tested positive for qPCR were found in all herds, but in significantly different proportions, from 13.4%  up to 56.2%  and 71.2% . High-shedding cows  or age at sampling (p = 0.22). In all instances, the herd random effect was highly significant (p < 10−6), indicating a strong herd effect on the probability of Map shedding. None of the logistic mixed regression models that were fitted indicated a significant effect on the odds of Map shedding of either days in milk within the lactation (p = 0.07).Overall, the probability of shedding was lower in vaccinated cows compared to non-vaccinated ones, but this result only approached statistical significance , when compared to non-vaccinated cows   A. Distinn = 137) , but not for those that were vaccinated after 4 months of age and/or tested positive at least once on ELISA (p > 0.5) (n = 20) were at higher risk to test positive with fecal qPCR compared to those that always tested negative (n = 245) . Finally, the best fit to the data was provided by models also accounting for the serological status. Indeed, a significant reduction in the odds of shedding was observed in cows vaccinated before 4 months of age and that tested negative for Map antibodies throughout the study period (p > 0.5) B. Once ap < 10−6), with Herds A and B associated with the lowest odd of a positive result and Herds F and G with the highest. A strong significant herd effect was found (−1 were considered positive (n = 72). Doubtful ELISA results did not influence model outputs and only the results where they were handled as positive are reported. Finally, these findings were still found when only samples with an estimated concentration of Map > 100·gn = 276). No difference in the estimated amount of Map shedding could be demonstrated between cows that always tested negative on serum ELISA and vaccinated cows, regardless of their age at vaccination or their serological status (p = 0.58). Conversely, non-vaccinated cows that tested positive on serum ELISA at least once had significantly higher shedding levels than all other cows (p < 10−4). It is noteworthy that high shedders  were observed in all groups (p = 0.006), whatever their vaccination status (p < 10−6), with Herds C and D associated with the lowest Map loads and Herds F and G with the highest. This analysis was performed on the subset of qPCR-positive fecal samples . We, therefore, sought to determine the factors influencing the variability of the serological response in vaccinated animals. The analysis was limited to the serological results obtained from the first sampling of each cow and was adjusted on age at vaccination, age at sampling, days in milk within the lactation, and the amount of Map shed in the contemporary fecal sample.p = 10−4), only age at vaccination had a significant effect (p = 0.002), with a higher probability of being positive in Map antibodies for cows that were vaccinated at an older age (p = 10−5).Taking into account the strong herd effect  cows reared under the same management conditions within each herd, this field study sought to evaluate vaccination as a management tool in paratuberculosis control programs. To the best of our knowledge, reports on the effect of Silirumlemented ,31,40,41lemented ,30, the lemented , and then = 10, 4.9%) or the vaccinated group . It was not a primary objective of the study to measure the effect of vaccination alone on the change in the herd prevalence of Map shedding, given that most herd farmers would use vaccination in combination with other disease control actions. This low infection rate may have reduced the possibility of evidencing a strong effect of vaccination, as would be expected in herds with high clinical incidence [Management changes aiming to reduce the transmission of Map were enacted long before the introduction of vaccination in all herds, and these changes have probably reduced the incidence of infection in young animals. In the year before the introduction of vaccination, the serological incidence in non-vaccinated cows was below 3% in 5 of the 7 herds, suggesting that the infection pressure may be low, as also supported by the low frequency of cows that seroconverted during the follow-up in the non-vaccinated control group (ncidence ,32,42,43ncidence ; with th® vaccine that it could last over the entire production life of animals [® vaccine. The fact that a few vaccinated cows may shed large amounts of Map in their feces, even among those testing negative for Map antibodies, is an argument for maintaining vaccination and good management practices for a very long period of time, even after clinical cases have disappeared, before a beneficial effect on Map transmission dynamics may be evidenced [Our findings that vaccinated cattle had detectable fecal shedding less often compared to controls contribute to the growing body of knowledge on the role of vaccination in achieving paratuberculosis control. However, it is well-established that vaccination does not guarantee the complete prevention of actual infection in animals, and that there exists a possibility of high shedding among vaccinated animals ,29,30,42 animals . This quvidenced ,47. A significant effect of the age at vaccination was observed, with a beneficial effect on the odd of Map shedding demonstrated only in cows vaccinated before 4 to 5 months of age. Our findings are in accordance with other studies ; the ageIn all models, a strong herd effect was evidenced, meaning that conclusions drawn on the overall effect of vaccination may not be valid in all herds. Interestingly Herds F and G were associated with both a higher probability of shedding and a higher amount of Map shedding in feces, even in vaccinated cows, suggesting that paratuberculosis control was less efficient in those herds. Common management practices designed to reduce Map transmission within each herd were monitored, and farmers were interviewed regularly to ensure that all the producers implemented them similarly, but departures from the best practices may have happened occasionally or repeatedly, which may explain different trajectories at the herd level . Others ® killed whole-cell vaccine, antibody response was still detected, as assessed by serum ELISA, up to 7 years after vaccination in some animals and, conversely, about one third of the vaccinated animals did not appear to develop a serological response [® vaccine, only 15% to 50% of sheep still tested positive on the serum ELISA 2 to 4 years post-vaccination, with large differences between flocks [® [Vaccination with either attenuated or inactivated paratuberculosis vaccines is known to induce a strong and long-lasting humoral response, but results vary widely according to the vaccine used or age at vaccination. Interindividual differences are also often reported. In a longitudinal field study involving calves vaccinated before 35 days of age with Mycoparresponse . In lambn flocks . In contn flocks . Interinlocks [® . Traditilocks [® ,55, but locks [® . In our locks [® ,57. Thislocks [® . Vaccinalocks [® ,59, whiclocks [® , to moreAltogether, our findings contribute to the growing body of knowledge on the role of vaccination in achieving paratuberculosis control, even in herds where good management and hygiene practices are already enacted. The fact that the beneficial effect of vaccination was only demonstrated in cows vaccinated before 4–5 months of age also provides practical information for the management of vaccination schedules in infected herds. Our results also reinforce the idea that even if beneficial in a few years, long-term vaccination is necessary to reduce the risk of new infections.Finally, non-specific effects of vaccination against paratuberculosis on lifespan have been suggested in cattle and goats ,49, whic


Lead (Pb) and cadmium (Cd) heavy metals are considered potentially hazardous toxins which cause serious health problems. Many studies reported that the water of Qaroun Lake in Fayoum, Egypt with its fish farms was contaminated with Pb and Cd above permissible levels. However, there is a lack of studies addressing levels of these toxic metals among inhabitants.We aimed to evaluate blood levels of Pb and Cd and their potential health risk among inhabitants around Qaroun Lake.This case-control study estimated Pb and Cd blood levels among 190 individuals from two destinations (near and far away) of Qaroun Lack using an atomic absorption spectrometer after full history taking and routine checkup investigations; Full blood count, serum ferritin, liver enzyme (ALT), and creatinine levels.There was a significant difference between blood levels of Pb and Cd heavy metals of inhabitants from near and far away Qaroun Lake destinations . The majority of inhabitants around Qaroun Lake had Pb and Cd blood levels above permissible levels (100% and 60% respectively). Critical levels out of them were 12.1% and 30.3% respectively. In comparison to inhabitants faraway Qaroun Lake, three individuals (2.4%) had Cd above the permissible level, while all of them (100%) had Pb level within the permissible level. There were no statistically significant differences between the two sampled populations as regards hemoglobin level, ALT, creatinine, and ferritin serum levels . The difference between studied populations regarding types of anemia was not statistically significant. Subclinical leucopenia was higher in the population near Qaroun Lake when compare to inhabitants far from the lake .Bio-monitoring of populations exposed to Pb and Cd hazardous substances could help in generating an early warning system to reduce the disease burden associated with their toxicity. Cadmium (Cd) and Lead (Pb) heavy metals, which are considered potentially hazardous toxins, cause serious health problems. Lead, cadmium, iron, and zinc were reported to be the commonest toxic metals that affect human health . Once a Pb is primarily toxic to the nervous system but can also induce anemia by disturbing the heme synthesis pathway . Cd whicPb and Cd heavy metals enter the environment from both industry and natural resources; such as volcanic emissions and weathering of rocks, therefore, they are found in air, soil, and water and can subsequently accumulate in plants and animals . In addithe Centers for Disease Control and Prevention (CDC), the average concentration of blood Pb among healthy unexposed adults was estimated at 0.9 ug/dL. The estimated reference values of permissible Pb blood levels are < 5ug/dL in adults and < 3.5 ug/dL in children, while the critical/ hazardous levels are ≥ 70 ug/dL in adults and ≥ 20 ug/dL in children (According to children . The avethe Joint Expert Committee on Food Additives (JECFA), an international scientific committee of the Food and Agriculture Organization of the United Nations in collaboration with the World Health Organization has established the safe level of provisional tolerable monthly intake (PTMI) of Cd and Pb heavy elements of 25 µg/kg of body weight  of Qaroun Lack as per the Declaration of Helsinki ethical standards and after taking the approval of the ethical committee of Faculty of Medicine Fayoum University Fig. . The samFull history taking and clinical examination that addressed the risk of exposure to Pb and Cd either environmental or occupational risk, as well as, any expected medical outcomes manifestation. Peripheral venous blood samples withdrew from each participant with informed consent into serum and ethylene diamine tetra acetic acid (EDTA) Becton Dickinson vacutainer tubes . Routine checkup investigations including Full blood count (CBC), serum ferritin, ALT liver enzyme, and serum creatinine levels were performed for the study population. Pb and Cd heavy metals were analyzed in serum samples of the participants using an atomic absorption spectrometer after sample separation and digestion using the ISO standard methods for determination of Pb and Cd levels.The sample digestion occurred depending on the ISO standard methods for wet digestion samples DIN EN 13805 . A weighAgilent atomic absorption spectrometer equipped by graphite furnace unit model 240ZAA  was used to determine cadmium (Cd) and lead (Pb) concentrations according to EN 14084:2003  was used for data statistical analysis. For lead and cadmium levels, median and interquartile range (IQR) were estimated. In comparing the two studied inhabitants, the Mann-Whitney U test was used. Categorical data were presented as frequencies and percentages. For comparing categorical data, the χ2 test or Fischer exact test was performed. P values less than 0.05 was considered to be statistically significant.The present study enrolled 190 residents aged (3–80 years) from two different districts located around Qaroun Lake in Fayoum Governorate in Egypt, where Ibshwai province located nearby the lake and Tamia province located far away from the lake were selected  was anemic, only less than one-fifth of them (14.3%) had chronic diseases with no statistically significant differences between the two studied populations   were iron deficiency anemia (IDA) confirmed with serum ferritin levels below normal cutoff value for gender and age. Additionally, subclinical leucopenia was higher in the population near Qaroun Lake when compare to those far from the lake  . The majority of inhabitants around Qaroun Lake had Pb and Cd blood levels much above permissible levels established by the CDC (100% and 60.6% respectively). Critical levels out of them were 12.1% and 30.3% respectively. In comparison to inhabitants faraway Qaroun Lake, three individuals (2.4%) had Cd above the permissible level, while all of them (100%) had Pb level within the permissible level  Table , howeverRegarding those inhabitants having microcytic hypochromic anemia with uncertain etiology, there was a statistically significant difference between them being higher among those living near Qaroun Lake with a p-value of 0.039 Table . Out of There is no data in the literature on the potential health risks of Pb and Cd heavy metals in the Egyptian population particularly in the region claimed to be potentially polluted, around Qaroun Lake in Egypt. Nevertheless, the previously published studies on the pollution of Lake Qaroun with heavy metals particularly Pb and Cd have been noted –18, 22. The historically renowned Qaroun Lake, a close ecosystem in nature, is located in the north of Fayoum Governorate in Egypt Fig. , where cGeorge and his colleagues (2013) reported that Cd concentration in the water samples collected from Wadi El-Rayan Lakes was within the recommended permissible limits (100 µg/L) of the National Environmental Law [Our study results coincide with previous studies in which Pb and Cd concentrations in water samples collected from Wadi EL-Rayan Lakes were higher than the maximum permissible level recommended by Egyptian Environmental law, (48) decision 1992/2013, in the spring/ hot season, but within permissible levels in cold one , 16, 22.ntal Law , where iIn our study, the median (IQR) blood concentration for Pb among our study population lived near Qaroun Lake was 36.25 (26.20–48.78) ug/dL, while it was 0 (0–1.7) ug/dL among those who lived far away the lake. Although various studies disclosed that the normal value for blood Pb concentration was 42.6 µg/L, different values were also reported for other countries. The mean Pb level in an Amazonian district was 32.77 µg/L, while it was 14.5 µg/L in Australia, 21.3 µg/L in Canada, 12.3 µg/L in the Americas, 65.4 µg/L in Brazil, 33.4 µg/L in Italy, and 19.1 µg/L in Korea . In concFurthermore, our results found that there were no significant differences between the population who lived nearby Qaroun Lake compared to those who lived far away the lack as regards age, gender, house, and occupation. However, we found that there was a significant difference between blood levels of Pb and Cd heavy elements of inhabitants from the two destinations around Quaron Lake . Inconsistent with previous studies described from Bangladesh , Mexico Queiroz and his colleagues (2019) reported that blood Pb levels show trends with age and gender being higher in adults and the elderly  blood levels and occurrence of noteworthy clinical/ subclinical manifestations in both kidney and liver functions when ALT, creatinine, and ferritin serum levels have been estimated in all the study population . The present study results, in agreement with previous studies, are considered an alarm to the toxic effect of environmental changes in Qaroun Lack ecosystem for being the final reservoir for most of such heavy metals, where it receives heavy loads of organic and inorganic pollutants via several agricultural drains and huge amounts of raw sewage, agricultural and industrial wastewater discharged into the lake , 47.Bio-monitoring of populations exposed to Pb and Cd hazardous substances could help in generating an early warning system to reduce the disease burden associated with their toxicity. However, a mass screening program for both inhabitants and environmental media of the regions nearby Qaroun Lake and Wadi El-Rayan in Fayoum Governorate is mandatory to validate such heavy metals’ hazardous impact.This study could be a preliminary study for bio-monitoring of the Fayoum population who are exposed to the hazardous effect of heavy metals in Lake Qarun water and its farm fish. Hence, to generate an early warning system to reduce the disease burden associated with their increased level and lethal toxicity; such as Alzheimer, kidney failure, and anemia for unknown causes, that are otherwise easily preventable.


This study compares the postoperative medical costs and outcomes of hip fracture patients treated with intravenous (IV) versus other analgesics .We performed a retrospective study at a tertiary hospital in Thailand, examining 1,531 patients who underwent hip fracture surgery between 2009 and 2020. We analyzed data on analgesic usage, costs, pain scores, and adverse effects.p = 0.004). Pain scores were similar across both groups, but the IV group had a significantly higher incidence of gastrointestinal side effects .In the study of 1531 patients, 63% of patients received as-needed analgesics, and 37% received preemptive prescriptions. In both groups, IV morphine was the predominant choice. The mean cost for the IV group was marginally higher than the other analgesics group ($2277 vs $2174). The other analgesics group had a significantly higher consumption of acetaminophen and selective NSAIDs (The choice of IV or other analgesics in treating hip fractures affects analgesic usage, side effects, medical costs, and patient outcomes. Further studies across different regions are recommended.The online version contains supplementary material available at 10.1186/s13018-023-04328-w. Among osteoporotic fractures, hip fractures are associated with significantly high morbidity and mortality rates as well as high medical costs –3. The hA key concern after hip surgery is postoperative pain because it is associated with delays in ambulation and prolonged hospital stays, as well as poor functional outcomes and reduced quality of life –12. TherOther non-opioid analgesics and modalities, however, come with a higher cost. Patients have been reported to typically spend about €7,500 for epidural analgesia compared to €7,273 for morphine patient-controlled analgesia (PCA) . SimilarIntravenous morphine is among the analgesics commonly used postoperatively, especially in hip fracture surgery. Morphine apparently has a greater ability to control severe pain compared to other analgesics . CurrentThe objective of this study was to compare IV morphine and other multimodal analgesics in terms of analgesic drug expense, length of hospital stay, postoperative complications, time to rehabilitation, pain score before and after rehabilitation, delay in ambulation, analgesic-related complications, over-and under-prescription of analgesics and functional outcomes in postoperative hip fracture patients including the types and quantities of analgesics used. The results of the study will hopefully promote discussion regarding the use and effectiveness of analgesics as well as providing support for additional safety precautions related to their prescription.This retrospective cohort study reviewed daily admissions to our center of patients with an intertrochanteric or femoral fracture from 1 January 2009 through 31 January 2020. A trained research associate collected patient’s data through a review of hospital medical records. Baseline characteristics of patients were obtained from reviews of medical charts.The inclusion criteria were age 18 years or older on admission and having undergone a primary inpatient surgical hip fracture procedure. The exclusion criteria were patients without a hip fracture or with no procedure codes for hip fracture surgery, patients with a previous hip fracture, patients with a length of hospital stay of more than 30 days, patients who used other strong opioids and other routes of morphine consumption, and patients who underwent a local nerve block during the hip operation. The patients were divided into two groups: patients who received intravenous morphine (the IV group) and patients who received the other analgesics group. Information was obtained on all analgesic drugs administered for each patient, including oral and injection medications as well as the use of transdermal patches.We determined the total IV morphine each patient consumed using medical records and chart reviews, both certified by a physician and a nurse. According to hospital policy, the usage of IV morphine is also monitored by requiring that any unused IV drugs be returned to the hospital and that the amounts returned be recorded. The quantity of IV morphine used was categorized into that used before surgery, post-operation, and recovery prior to rehabilitation. We also recorded the frequency of use separately to help confirm the total consumption and to calculate the average IV morphine dosage. Notes on anesthesia use and patient medical records provided patient body weight information. We calculated the per kilogram dose by dividing the total amount used by the patient's body weight. To calculate postoperative morphine use, the quantity of postoperative IV morphine used was divided by body mass since the surgery was conducted after the first rehabilitation period.All other analgesics used for each patient were tracked using the hospital's analgesic drug lists for four categories of analgesic drugs: acetaminophen, non-selective NSAIDs, selective NSAIDs, and weak opioids. Each category of drug was then separated into oral drugs and IV drugs. The total consumption, total cost, and cost per unit of each of the analgesic drugs was recorded. The per unit cost by weight of each drug was used to calculate the total cost of drugs used with each patient.Three routes are available for analgesic drug administration: oral, injection, and patch form. The oral route was the standard used for pain control while injection was used in cases of 99%. As the patch was used with only a small minority of patients, data on patch delivery was recorded but was not used in the comparison of the two study groups. Analgesic drug usage and cost in the IV and other analgesics groups were also collected and compared.We examined the relationship between the level of pain in the IV and other analgesics groups as well as in-hospital outcomes, including total hospital cost, analgesic drug cost, length of stay, postoperative complications, time to rehabilitation, pain score before and after rehabilitation, delay in ambulation, analgesic complications, and functional outcomes. Based on the data available in hospital records, patients' level of pain was calculated as a numeric score on a scale of 1 to 10, where NS 1 = no pain and NS 10 = very severe pain. Pain scores were determined immediately after a patient was transferred to the ward following surgery then every 4 h until entry into the the rehabilitation program. Patients with moderate (NS 5-7) to severe pain (NS > 7) were identified and their analgesic drug use information was recorded. Physicians independently reviewed each patient's complications which had been recorded at the initial visit plus the dosages of antiemetic drugs following the International Statistical Classification of Diseases and Related Health Problems (ICD10).PubMed and Scopus, include problems involving the respiratory system, gastrointestinal system, central nervous system and genitourinary system, as well as other adverse effects such as bradycardia, rash, itching, and falls from bed. Each possible adverse effect was matched with ICD-10 and the patient's history in the medical record following the hip fracture event. All the adverse effects are collected before using therapeutic drugs such as PPIs, antiemetics, and antibiotics drugs.Potential adverse effects of analgesic drugs, identified from Analgesic drugs used for each patient, including name, type, total cost, unit price, and total consumption, were obtained from the hospital records database. The number of drugs and the total cost of the drugs were recorded. The unit price of each analgesic drug was recorded at the time of use as some prices change over time. Trends in use of individual drugs were plotted to identify changes over time.p < 0.05. Statistical analysis was performed using STATA version 16  and Microsoft Excel .Patient analgesic use was divided into two groups: analgesics prescribed as needed and those prescribed preemptively using a pain score cut point of 3. Selected patient characteristics were used in the analysis for weighted adjustment, including actual age, age > 70 years, gender, BMI, body weight, height, type of fracture, type of operation, operation time, and type of anesthesia. Hospital costs, quantity, and cost of analgesic drugs were calculated and categorized by group. The heterogeneity of unweighted and weighted groups were defined by absolute standardized difference. Differences in continuous variables between the 2 groups were assessed using weight median regression; differences between categorical variables were determined using weight risk difference regression. Associations were considered statistically significant if A total of 1,531 patients age 18 years or older underwent primary hip fracture surgery in a tertiary-level medical school hospital in Thailand between 1 January 2009 and 31 January 2020. Characteristics of the patients are presented in Table The mean daily dose of IV morphine was 12 mg (IQR 9-18) in the prescribed as needed group and 9 mg (IQR 4-15) in the preemptive group. The mean dose of IV morphine in the first 24 h postoperatively was 12 mg (IQR6-15) in the prescribed as needed group and 7 mg (IQR 3-12) in the preemptive group. In comparison, the mean postoperative dose of IV morphine before rehabilitation was 12 mg (IQR 8-18) in the prescribed as needed group and 9 (IQR 4-15) in the preemptive group. The mean single dose of IV morphine 24 h postoperatively was 0.20 mg/kg (IQR 0.12–0.30) in the prescribed as-needed for pain control group. The overall mean cost of IV morphine per patient was 7.2 baht (IQR 5.4-10.8) . Mean diagnostic and radiological treatment was 1500 baht (IQR 1100–2200) and 1760 baht (IQR 1320–3150) (p = 0.026) in the other analgesics group and the IV morphine group, respectively. In the preemptive group, the average cost of room and food cost was statistically significantly different between the two groups: 10,000 baht  in the other analgesics group and 7800 baht  in the IV morphine group. Other costs showed no significant difference between the groups.The hospital costs of inpatient hip fracture treatment for each service unit are recorded in Tables p = 0.022), but not in the mean amount: (30.5 baht (IQR 18–52) in the other analgesics group and 20 baht (IQR10-40) in the IV morphine group (p = 0.19). The mean amount of acetaminophen consumed in the analgesics prescribed as needed group was statistically significantly higher in the other analgesics group than in the IV morphine group: 100 mg (IQR 64–120) and 82 mg (IQR 64–120) (p = 0.004), respectively. The difference in the mean cost of acetaminophen in the two groups was also statistically significantly different at 50 baht (IQR 32–70) and 46 baht (IQR 32–60), respectively.The amount and cost of each analgesic drug in the two groups are shown in Tables Additional file p < 0.01): 91 (22.14%) and 14 (8.92%) in the IV morphine group and other analgesics group, respectively.Table This study investigated differences between IV morphine consumption and other analgesics consumption patients in terms of hospital costs, analgesic drug expense, postoperative complications, adverse analgesic effects, and functional outcomes. The participants in each group were subdivided into an as needed-for-pain control group (pain score ≥ 3 points) and a preemptive analgesic use group.Patients prescribed IV morphine as-needed for pain control exhibited significantly higher total hospital, diagnostic, radiological treatment, and home medical fee costs than the other analgesic group. Both preemptive analgesic sub-groups demonstrated statistically significant differences only in room and food costs. In contrast, total hospital costs were notably higher in the as-needed IV morphine group, with a mean cost of approximately 75,898 THB , respectively. Notably, the cost of diagnostics, radiological treatment, room and food, and home medication fees were significantly higher in the as-needed IV morphine group. Globally, the pooled mean cost for the index hospitalization was US$10,075   , with thIn this study, the cost of diagnostics, radiological treatment, room and food, and home medication fees were significantly higher in the IV morphine were prescribed as-needed for pain control group than the other analgesics. Factors that could potentially explain these results include higher demands on services related to pain and its side effects in the IV morphine group and the limitation of analgesic choices regarding the patients’ health coverage package. The cost of home medication was also higher in the other analgesics group because that group received more oral drugs for pain control. Costs for inpatient hip fracture treatment can be incurred in the areas of acute stay, operative treatment, laboratory investigation, and radiology. Acute stay costs, including nursing service, room, and food, are the main factors, accounting for about 84% of total inpatient costs . HoweverNo difference was found between IV and other analgesics patients in the use of all analgesic drugs in the preemptive drug group or in the use of other drugs, including combination drugs, non-selective NSAIDS, and weak opioids for controlling pain. The difference in the consumption and cost of acetaminophen and selective NSAIDs identified in this study is a reflection of a limitation of the government health care coverage system in Thailand. Currently, the most widely used public health protection schemes available to all Thai citizens are the Universal Health-care Coverage Scheme (UCS) which is available to about 76% of the population followed by the Social Security Scheme (SSS) at 15%. In-patient services are reimbursed using a case-mixed system, Diagnostic Related Groups (DRG). These systems limit the amount of money paid to hospitals , 28.Gastrointestinal, respiratory, and genitourinary-related complications are the most frequent adverse events associated with orthopedic procedures . GI sideThis study found only a limited association between potential risk factors and immediate postoperative pain after hip fracture surgery, unlike a previous study which reported lower education level (< 8 yrs of school) and the presence of psychological depression as being significantly associated with severe postoperative pain . A detaiTreatment of hip fractures with either intravenous morphine or other analgesics appears to be associated with increased use of NSAIDs and/or acetaminophen as well as the incidence of GI side effects.The decision to treat with either intravenous or other analgesics may also significantly affect medical expenses and hospitalisation outcomes. Additional studies of these relationships are needed, especially in different geographic and political settings.Additional file 1. Average pain score over time.Additional file 2. Table of morphine comsumption.


Self-emulsifying drug delivery systems (SEDDSs) are lipid-based systems that are superior to other lipid-based oral drug delivery systems in terms of providing drug protection against the gastrointestinal (GI) environment, inhibition of drug efflux as mediated by P-glycoprotein, enhanced lymphatic drug uptake, improved control over plasma concentration profiles of drugs, enhanced stability, and drug loading efficiency. Interest in dermal spontaneous emulsions has increased, given that systems have been reported to deliver drugs across mucus membranes, as well as the outermost layer of the skin into the underlying layers. The background and development of a double spontaneous emulsion incorporating four anti-tubercular drugs, clofazimine (CFZ), isoniazid (INH), pyrazinamide (PZY), and rifampicin (RIF), are described here. Our methods involved examination of oil miscibility, the construction of pseudoternary phase diagrams, the determination of self-emulsification performance and the emulsion stability index of primary emulsions (PEs), solubility, and isothermal micro calorimetry compatibility and examination of emulsions via microscopy. Overall, the potential of self-double-emulsifying drug delivery systems (SDEDDSs) as a dermal drug delivery vehicle is now demonstrated. The key to success here is the conduct of preformulation studies to enable the development of dermal SDEDDSs. To our knowledge, this work represents the first successful example of the production of SDEDDSs capable of incorporating four individual drugs. Lipid-based drug delivery systems are under intensive examination for improving the oral bioavailability of lipophilic drugs ,2. Self-The mucus gel layer of the GI tract comprises water 90–95%), glycoproteins, and additional minor components such as DNA, lipids, and electrolytes %, glycop. Hence, Compared to conventional drug administration routes, the dermal route is considered safe and non-invasive, and is especially useful for delivering drugs of poor aqueous solubility . Two impn = 3) values for the different oils tested. No external oil phase mixed with beeswax or linseed oil displayed clear separation. Further, no separation was detected between shea butter and any internal oil phase. Therefore, Transcutol®:PEG (9:1) was selected as the only suitable internal oil phase. Clear separation between Transcutol®:PEG (9:1) and natural oils was detected in the following order: evening primrose oil (EPO) > olive oil (OLV) > avocado oil (AVO) > palm oil (PALM) > safflower oil (SAFF).All experiments were carried out in triplicate, and the results are expressed in ® and PEG due to their relatively high dielectric constants and hydrogen bonding [® also known as diethylene glycol monoethyl ether, is a liquid well known to the cosmetic industry due to its favorable solubility properties that improve the solubilization of lipophilic and hydrophilic drugs [® is frequently included as a co-surfactant and skin penetration enhancer [® to a dermal formulation with an aqueous component will influence the ionization equilibrium of the incorporated drugs, should they be capable of undergoing ionization [® will increase the quantity of un-ionized drug able to cross the skin barrier [These results can be explained by the polar natures of Transcutol bonding ,12. Tranic drugs . Moreoveenhancer . The add® in dermal formulations is advantageous, as the absence of water decreases the thermodynamic driving force needed to enable penetration of drug into the skin [® to fulfil the role of the external oil phase. Clear phase separation of water-in-oil-in-oil (W/O/O) emulsions will potentially be exhibited due to the hydrophilic nature of Transcutol®–PEG mixtures. However, because addition of water and the formation of binary mixtures of Transcutol® with water favor dermal drug delivery, compared to Transcutol® alone it would be inappropriate to include the Transcutol®–PEG mixture as the external oil phase and separate water and Transcutol® with an internal oil phase. This may prevent the SDEDDSs from releasing the incorporated drugs, despite exhibiting desirable SDEDDSs properties [® is considered a well-tolerated topical excipient with the ability to solubilize both hydrophilic and lipophilic drugs, which may be highly beneficial when considering the various lipophilicity profiles of model drugs [It is reported that inclusion of both water and Transcutolthe skin . The addthe skin . This isthe skin ,6. Henceoperties . Moreoveel drugs .® to solubilize both lipophilic and hydrophilic drugs [®, and the inclusion of hydrophilic drugs such as isoniazid (INH) and pyrazinamide (PZY) can benefit from the presence of a greater aqueous component [A large self-emulsification area was observed, which may signify appropriate excipient combinations. As indicated in ic drugs . Furtheromponent .As indicated in Rapid emulsification is highly favorable when developing oral SEDDSs or SDEDDSs, as spontaneous emulsification is a rate-limiting step preceding successful drug absorption . HoweverThe PEs were visually inspected after removal from a water bath set at a temperature of 68 °C for a period of 3 h . No phasThe poor robustness shown by sudden short exposure to a high temperature may be associated with an increase in the surfactant concentration. Interestingly, an elevation of the surfactant content can reduce the size of the droplets until the surfactant concentration reaches a concentration at which the size of the droplets increases, as established by the aggregates produced from excess surfactant included . The sud®:PEG together with surfactant. It may seem to be unusual to use the fixed combination of the internal oil phase and the surfactant phase as a single component of the triplot instead of plotting the water and the internal phase together, while plotting the surfactant phase separately. However, the use of decreased surfactant and internal oil phase concentrations are imperative, as the propensity for self-emulsification is intensified with limited surfactant phase content. Decreased surfactant concentrations imply a reduced risk of skin irritation, especially during prolonged use, as well as lowered production costs [®–PEG content of SDEDDSs, as Transcutol® should be included in moderate amounts compared to water, due to its high affinity for water that limits the dermal diffusion if it is not accompanied by sufficient water concentrations [on costs ,6. Moreotrations .®:PEG:surfactant phase ratios. This phenomenon is attributed to the instability of SDEDDSs due to increased surfactant concentrations [® can also be employed as a co-surfactant [®:PEG:surfactant phase is not optimized, Transcutol® can enhance the effect of the surfactant phase and thus further contribute to the emulsion instability. The gray areas indicated in the 7:4 ratio  in the pseudoternary phase diagrams signify that self-emulsification was observed, but the consistency of the SDEDDSs changed to high viscosity, eliciting poor formulation properties such as creaming and excipient precipitation. Again, this is a clear indication that the use of increased concentrations of the surfactant phase are not favorable. Interestingly, this can be related to the reduced difference in the ratio of the internal oil phase compared to the surfactant phase, represented by the 8:3 and 7:4 ratios . Therefore, it was decided to maintain an internal oil-phase-to-surfactant-phase ratio of 9:2.Excellent self-emulsification was exhibited during the construction of the pseudoternary phase diagrams of the tested SDEDDSs, as seen in trations . It shourfactant . Therefon = 3).Solubility studies are crucial for determining the amount of excipients needed to best solubilize different drug combinations, while maintaining concentrations close to saturation to enhance the driving force needed to achieve dermal diffusion ,6,19. Th® and the surfactant phase to the water phase. This addition improved the aqueous solubility of all drugs, since Transcutol® can solubilize both lipophilic and hydrophilic compounds [As indicated, the lipophilic drugs, CFZ and RIF, displayed a higher solubility in natural oils compared to the more hydrophilic drugs, INH and PZY. Moreover, INH and PZY exhibited increased solubility in the PE. As expected, the aqueous solubility of individual drugs indicated in ompounds . Similarompounds . ClearlyPALM induced the highest solubilization of CFZ, whereas RIF had the highest solubility in AVO. However, as CFZ causes dose-dependent skin discoloration, the aim is to reduce the concentration of CFZ when it is used together with PZY. It has been found that the dose of CFZ can be substantially reduced from 25 mg/kg to 6.25 mg/kg when administered orally together with PZY due to the synergistic activity achieved when CFZ and PZY are used in combination . Hence, Isothermal microcalorimetric studies indicated that differences existed between the slopes and y-intercepts of the measured and theoretical (no interaction) heat flows of certain samples tested during compatibility studies. Additionally, the absolute values of the interactions integral to these samples were also greater than zero. Therefore, potential interactions were observed for the following combinations: OLV and PE (−38.01 J/g); AVO and PE (2.64 J/g); EPO and PE (−29.18 J/g); PALM and PE (26.20 J/g); SAFF and PE (85.90 J/g); PE and PZY (−26.54 J/g); PE and CFZ (36.36 J/g); PE and RIF (39.15 J/g); PE and INH (36.61 J/g); CFZ and INH (2252 kJ/g); as well as INH and RIF (822.89 J/g). However, potential interactions between PE and selected external oil phases were expected because the external oil phases were included due to the clear immiscibility between the internal oil phase and external oil phases during the oil immiscibility studies. Therefore, this expected so-called incompatibility is only due to the inability of the oils to mix, and is considered insignificant, as these excipients were intentionally included as a result of their immiscibility with the internal oil phase in order to enable production of double emulsions.®83, Transcutol®, Tween®60 and water, excipients that are frequently included in dermal formulations due to their favorable safety, solubilizing, and emulsion-stabilizing properties [Remarkably, all of the model drugs exhibited potential incompatibility with the PE. Nonetheless, this may be explained by the increased solubility of the drugs observed in the PE compared to the aqueous solubility reported in the literature. The PE comprises Spanoperties ,6,12. ThIn terms of drug–drug interactions, it is noted that synergism is obtained when CFZ is used in combination with the first-line antitubercular agents INH and RIF . This coEvaluation of the PEs by microscopic examination revealed that the PE ratio of 9:9:2  was the only PE that exhibited droplet formation. This supports the decision to exclusively use this ratio, and only vary the external oil phase according to the antitubercular drugs included in the SDEDDSs. The microscopic image was captured without any drugs present, as shown in n = 3). The droplet size was measured as an average of 173.90 ± 2,30 nm and a PDI value of 0.236 ± 0,11. As shown in ®83 and Tween®60 included as surfactants may also contribute to the ability of the PE to remain stable, since PEG can be used as a surface-active agent as well as a solvent [PE 1 (9:9:2) was subjected to droplet size analysis and polydispersity index (PDI) determination. The results are expressed as mean ± SD  by means of gentle stirring until fully combined. At the same time, the surfactant phase was separately prepared by gently stirring Span®60 and Tween®83 in a ratio of 1:1 until completely blended. Next, surfactant phase and the internal oil phase were combined  while being subjected to mild stirring for a duration of 30 min. Thereafter, a predetermined quantity of water, a ratio of 9 in this case, was added in small increments while applying steady agitation. After subjecting this mixture to homogenization for 4 min, in order to generate a PE, the pre-determined quantity of five parts external oil phase was added in a dropwise fashion while gently stirring. After continuous stirring, the mixture was removed from the stirring plate and subjected to sonication until the desired droplet size was achieved. Drugs were included at a concentration of 2% each. Hence, overall composition of SDEDDS can be summarized as follows: 8% drugs (2% of each drug), 36% water, 20% AVO, 29.5% internal oil phase, and 6.5% surfactant phase.The checkpoint formulation, namely 9:9:5  to outer oil phase) was prepared. Preparation was commenced by mixing internal oil phase components (Transcutol(n = 3). Average droplet size was revealed to be 92.46 ± 5.52 nm, and the PDI value found to be 0.391 ± 0.05. Therefore, the selected PE and SDEDDS were visually examined via transmission electron microscopy (TEM) for the purpose of gaining a reliable visual representation of the droplets that fell into the nano size range. As the highlight of this study, the TEM observation revealed clear small droplet formation inside larger smooth-surfaced droplets, signifying the successful development of a SDEDDS designed to simultaneously contain four drugs. Moreover, the mechanism of spontaneous emulsification was captured as indicated in The checkpoint SDEDDS was characterized by measuring droplet size and PDI. The results are expressed as mean ± SD As indicated in As seen in ®, Span®83, and Tween®60 were purchased from Merck . AVO, beeswax, EPO, linseed oil, OLV, PALM, SAFF, and shea butter were bought from Nautica Organics Trading . PEG was obtained from DB Fine Chemicals . Distilled water was acquired through a Rephile Bioscience Ltd. system .Combinations of CFZ, INH, PZY, and RIF were utilized as model drugs. CFZ, INH, and RIF were kind gifts from Prof Wilna Liebenberg, head of the Solid-State Pharmaceutical Innovation and Nanotechnology (SPIN) research group at the North-West University . PZY, TranscutolAntitubercular drugs were selected on the basis of their physicochemical properties and Biopharmaceutical Classification System (BCS) ranking, as indicated in From Mycobacterium tuberculosis [Mycobacterium tuberculosis that might lead to drug resistance [Developing a topical dosage form containing four drugs to aid in cutaneous tuberculosis (CTB) disease is challenging but necessary due to the daunting nature of rculosis . Hence, sistance ,37. Hencsistance . Howeversistance .Mybobacterium fortuitum and Mycobacterium abscessus complex together with other agents such as amikacin, trimethoprim–sulfamethoxazole, linezolid, tetracyclines, quinolones, and gepotidacin (for Mycobacterium fortuitum complex) and tigecycline, clarithromycin, omadacycline, and thiostrepton (for Mycobacterium abcessus complex) [CFZ, a riminophenazine antibiotic, is listed by the World Health Organization as a Category B agent for treatment of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) . A meta-complex) . Unfortucomplex) . Howevercomplex) .It was decided to formulate a W/O/O emulsion for the purpose of providing a water phase to optimally solubilize the hydrophilic drugs, and oil phases capable of ensuring effective solubilization of the lipophilic drugs. Ideally, the external oil phase should possess additional skin penetration enhancement properties to facilitate dermal drug delivery . However®:PEG (9:1 ratio), with a combined HLB value of 4.46, were analyzed to find an internal oil phase [Beeswax , linseed oil  and Transcutolil phase ,48,49. S®83  and Tween®60  were selected as surfactant and co-surfactant, respectively [®83:Tween®60 (1:1) was maintained throughout the experiments, as this ratio has demonstrated greater stabilization of SEDDSs compared to higher ratios that increase the emulsion range but reduce stability, as indicated by drug precipitation [Choosing the correct surface active agent(s) is essential in order to stabilize the emulsions once formulated . The HLBectively ,56. A fipitation .®:PEG (9:1), water and the surfactant phase  were combined in different ratios to display a triplot. This then is able to indicate the most appropriate combination of additives required to form successful SDEDDSs, as one can establish the self-emulsifying areas and also validate the most effective concentrations and ratios of the included composites. No drugs were used, as it was necessary to establish excipient behavior without the influence of any drugs. The surfactant phase and water were mixed in fixed ratios , while Transcutol®:PEG (9:1) was added in a dropwise manner as a variable component at an ambient temperature. The concentration at which the turbidity of the mixtures is first observed was plotted as the endpoint, employing Triplot software version 4.1.2 [A water titration method was utilized to construct a pseudoternary phase diagram wherein Transcutolon 4.1.2 . Thus, t®:PG; 9:1) to established quantities of the water and surfactant phases. The mixtures were then subjected to homogenization with a Heidolph DIAX600 homogenizer  for 4 min at 9500 rpm, while being kept at room temperature [The PEs selected from the pseudoternary phase diagram were subsequently prepared by adding known quantities of the internal oil phase  with a clear or bluish color;Grade B: Fast-forming (within 1 min) and somewhat less clear emulsion with a bluish-white color;Grade C: Fine murky emulsion that forms within 2 min;Grade D: Dull, grayish-white emulsion that displays a slightly oily appearance, which indicates slow emulsification (longer than 2 min);Grade E: A formulation demonstrating either poor or minimal emulsification with large oil droplets existing on the surface.Complete emulsification and emulsification time can be defined as the time required to obtain a macroscopically homogeneous emulsion . To obtaEmulsions graded as C or grade D were deemed favorable for dermal drug delivery . For thiThe PEs (10 mL) were placed in a water bath set at a temperature of 68 °C for a period of 3 h prior to evaluation . After tThe water titration method was again utilized to construct pseudoternary phase diagrams. Drugs were again excluded during pseudoternary phase diagram construction to be able to observe excipient response without the complication of variables introduced by drug inclusion. The internal oil phase was mixed together with the surfactant phase  and water in fixed proportions , while individual external oil phases were added dropwise as the variable component at an ambient temperature. The concentration at which the turbidity of the mixtures is observed was plotted as the endpoint, employing Triplot software version 4.1.2 ,62,63.® filter and diluted with methanol [An excess amount of individual antitubercular drugs was added to 5 mL of the different vehicles considered during this study. Samples were then vortexed for approximately 2 min. Solubility studies were performed by placing samples in the circular axis (54 rpm) of a rotating solubility bath set at 32 °C (±0.5 °C) for a period of 48 h . The sammethanol . A validmethanol .The compatibility of excipients and drugs was investigated using a previously published method . A 2277 All visual examinations of either the PEs or the SDEDDSs were conducted utilizing an Olympus microscope . TEM measurements were performed on a TECNAI G2 (ACI) instrument operated at an accelerating voltage of 200 kV  ,66.® ZS  at 25 °C [Droplet size and size distribution were assessed by means of dynamic light scattering performed using a Malvern Zetasizer Nanoat 25 °C . All samFuture in vitro drug release and permeation studies should be conducted in order to establish the release of the drugs from SDEDDSs. Additionally, nanotoxicology studies should also be performed for the purpose of establishing the extent as well as the safety of the permeation achieved by N-SDEDDSs . However