The Impact of Early Brain-Dead Donor Detection in the Emergency Department on the Organ Donation Process in Iran.

We aimed to assess the impact of hospital characteristics on the outcomes of detected possible brain-dead donors, in our organ procurement network in Iran. Data was collected through twice-daily calls with 57 hospitals' intensive care units and emergency departments over 1 year. The donation team got involved when there was suspicion of brain death before the hospital officially declared it. The data was categorized by hospital size, presence of neurosurgery/trauma departments, ownership, and referral site. Out of 813 possible donors, 315 were declared brain dead, and 203 were eligible for donation. After conducting family interviews (consent rate: 62.2%), 102 eligible donors became actual donors (conversion rate: 50.2%). While hospital ownership and the presence of trauma/neurosurgery care did not affect donation, early referral from the emergency department had a positive effect. Therefore, we strongly recommend prioritizing possible donor identification in emergency rooms and involving the organ donation team as early as possible. The use of twice-daily calls for donor identification likely contributed to the consistency in donation rates across hospitals, as this approach involves the donation team earlier and mitigates the impact of hospital characteristics. Early detection of possible donors from the emergency department is crucial in improving donation rates.

Abdominal pain in a young lady with inverted Meckel's diverticulum: a case report.

Meckel diverticulum is the most common congenital anomaly of the gastrointestinal tract which is located in small bowel within 2 feet of the ileocecal valve. Nevertheless, an inverted Meckel's diverticulum is an uncommon condition believed to result from aberrant peristalsis in that specific area. This article showed signs, symptoms, and possible clinical presentations using CARE guidelines in a case of inverted Meckel's diverticulum and reviews other possible features lastly, definitive treatment, results, and case follow-up were shown to refresh, and raise surgeons' awareness of this rare disorder.

Facilitators and barriers in the donor family interview process from the perspective of hospital staff: a cross-sectional study.

Background: Obtaining consent from potential donor families is a challenging step in the donation process and is influenced by various factors. Methods: In this cross-sectional study, we utilized a questionnaire containing 14 questions about facilitators and barriers in the family interview process. The questionnaire was distributed in March 2023 to intensive care unit (ICU) nurses who had experience with donor family interviews. We collected the opinions of these respondents on hospital performance and drew comparisons between the studied hospitals. Results: A total of 60 participating ICU nurses provided mean scores for hospital performance in family interviews of 2.60±0.84 for type I hospitals (those providing neurosurgery and trauma care) and 2.035±0.890 for type II hospitals (those without neurosurgery and trauma services; P=0.04). The mean scores for public and private hospitals were 1.86±0.86 and 2.59±0.85, respectively (P=0.008). Based on the findings, the most important facilitators were the availability of organ donation staff and access to a professional team for family discussions. Conversely, poor physician communication skills and limited communication capabilities among medical staff were identified as significant barriers. Implementation of a professional team for family interviews was found to be more critical for type II hospitals. Poor physician communication skills were a significant concern in public hospitals, while families' lack of awareness of patient prognosis emerged as a key barrier in private hospitals. Conclusions: This study highlights numerous facilitators and barriers that vary across hospitals. Addressing these issues individually and developing tailored plans to enhance hospital performance in interviewing donor families is essential.

Successful Surgical Management of Ruptured Hepatic Artery Pseudoaneurysm Post-Liver Retransplant: A Case Report.

INTRODUCTION: Hepatic Artery Pseudoaneurysm (HAP) is a rare and potentially fatal complication following liver transplantation, with unclear etiology. Various predisposing factors, including local infections, biliary-enteric anastomosis, particularly Roux-en-Y hepaticojejunostomy, vascular interventions, and iatrogenic injuries, have been identified. PRESENTATION OF CASE: We report a case of HAP occurring after liver re-transplantation in a twenty-year-old male patient who had initially undergone liver transplantation for cirrhosis resulting from autoimmune hepatitis. Subsequently diagnosed with chronic ischemic liver disease accompanied by diffuse cholangiopathy and a liver abscess, the patient underwent a liver re-transplantation. Within the early days post-re-transplantation, the patient presented symptoms of gastrointestinal bleeding and sudden hypotension. A high clinical suspicion led to prompt laparotomy, ultimately successful preserving both the graft and the recipient's life. DISCUSSION: HAP is an uncommon complication following liver transplantation. This condition may remain asymptomatic until the rupture of the HAP, which can result in gastrointestinal bleeding, hypotension, and, eventually, mortality. While there have been emerging non-surgical techniques proposed for HAP treatment, surgical intervention remains the definitive solution for preserving the graft and ensuring the recipient's survival. CONCLUSION: HAP, while uncommon, represents a life-threatening complication post-transplantation. In the event of rupture, immediate intervention is imperative for the patient's survival. Hence, it is critical for surgeons to be cautious about this complication, recognize its symptoms, and be aware of associated risk factors.

Surgical Interventions in Coronavirus Disease 2019 Pleural and Pulmonary Complications: A Case Series of the Tertiary Thoracic Surgery Center Experience.

Background: In most cases of pulmonary or pleural post-coronavirus disease 2019 (COVID-19) complications, surgical interventions are performed to treat these complications, but the method of the surgery and its outcome in these patients is not clearly defined. We present 40 patients with pulmonary and pleural complications after COVID-19 who required surgical intervention. Patients and Methods: In this case series, patients' data were prospectively collected from April to August 2022 at Masih Daneshvari Hospital. Inclusion criteria were patients with COVID-19 who were referred to the thoracic surgery department because of pleural effusion, pneumothorax, empyema, infected or non-infected pneumatocele, and lung cavity with suspected fungal infections. The required intervention for each patient was assessed. Results: Patients' mean age was 49.21 ± 11.5 (30-69 years). Nine patients (22.5%) were female. Pure pleural effusion was reported in five (12.5 %), pneumothorax in eight (20%), empyema in 29 (72.5%), and infected pneumatocele in two patients (5%). Twelve patients had bronchial fistulas that were clarified at the time of surgery that needed repair after resection. In 13 patients (32.5%) because of pleural effusion or pneumothorax, a chest tube was inserted and after two weeks lungs were fully expanded. All patients with pneumothorax were managed by chest tube initially but in the presence of continuous air leakage and non-expanding lungs surgical thoracotomy or video-assisted thoracoscopic surgery (VATS) were considered for correction. In 10 patients who required thoracotomy, the chest tube was necessary for more than one month. In most of the patients, there were small cystic lesions or peripheral bronchopleural fistula. In 17 (42.5%) cases of empyema, necrotic pneumonia was documented and eight patients (20%) had aspergillus infection in the pathology report and two patients had a pulmonary abscess. Conclusions: Pleural COVID-19 complications can be treated with conventional surgical methods such as chest tube insertion, and debridement of infected tissue with no mortality and further complications.

Brain Death Confirmation by 18F-FDG PET/CT: A Case Series.

OBJECTIVES: Brain death is a state of irreversible loss of brain function in the cortex and brainstem. Diagnosis of brain death is established by clinical assessments of cranial nerves and apnea tests. Different conditions can mimic brain death. In addition, confirmatory tests may be falsely positive in some cases. In this study, we aimed to evaluate the role of positron emission tomography-computed tomography scan with 2-deoxy-2[18F]fluoro-D-glucose (18F-FDG-PET/CT) as an ancillary test in diagnosing brain death. MATERIALS AND METHODS: We analyzed 6 potential brain death donors for the confirmatory diagnosis of brain death using FDG-PET/CT. All 6 donors were brain dead by clinical criteria. All patients had electroencephalogram and brain computed tomography. Other than FDG-PET/CT, transcranial Doppler was performed in 1 patient, with other patients having no confirmatory ancillary imaging tests. Patients had nothing by mouth for 6 hours before imaging. Patients were supine in a semi-dark, noiseless, and odorless room with closed eyes. After 60 minutes of uptake,the brain PET/CT scan was performed with sequential time-of-flight-PET/CT (Discovery 690 PET/CT with 64 slices, GE Healthcare). The PET scan consisted of LYSO (Lu1.8Y0.2 SiO5) crystals with dimensions of 4.2 × 6.3 × 25 mm3. Three-dimension images were with scan duration of 10 minutes. RESULTS: The PET scan confirmed brain death in 5 of the 6 cases. However, we ruled out brain death using PET/CT in a 3-year-old child, although all clinical tests confirmed brain death. CONCLUSIONS: A PET scan illustrates a hollow skull phenomenon suggestive of brain death. It can be a powerful diagnostic tool to assess brain death.

Reoperation etiologies in the initial hospital stay after liver transplantation: a single-center study from Iran.

Background: Liver transplantation (LT) is widely recognized as a life-saving therapy for patients with end-stage liver disease. However, due to certain posttransplant complications, reoperations or endovascular interventions may be necessary to improve patient outcomes. This study was conducted to examine reasons for reoperation during the initial hospital stay following LT and to identify its predictive factors. Methods: We evaluated the incidence and etiology of reoperation in 133 patients who underwent LT from brain-dead donors over a 9-year period based on our experiences. Results: A total of 52 reoperations were performed for 29 patients, with 17 patients requiring one reoperation, seven requiring two, three requiring three, one requiring four, and one requiring eight. Four patients underwent liver retransplantation. The most common cause of reoperation was intra-abdominal bleeding. Hypofibrinogenemia was identified as the sole predisposing factor for bleeding. Frequencies of comorbidities such as diabetes mellitus and hypertension did not differ significantly between groups. Among patients who underwent reoperation due to bleeding, the mean plasma fibrinogen level was 180.33±68.21 mg/dL, while among reoperated patients without bleeding, it was 240.62±105.14 mg/dL (P=0.045; standard mean difference, 0.61; 95% confidence interval, 0.19-1.03). The initial hospital stay was significantly longer for the reoperated group (47.5±15.5 days) than for the non-reoperated group (22.5±5.5 days). Conclusions: Meticulous pretransplant assessment and postoperative care are essential for the early identification of predisposing factors and posttransplant complications. In order to enhance graft and patient outcomes, any complications should be addressed without hesitation, and appropriate intervention or surgery should not be delayed.

Effectiveness of different vaccine platforms in reducing mortality and length of ICU stay in severe and critical cases of COVID-19 in the Omicron variant era: A national cohort study in Iran.

Various severe acute respiratory syndrome coronavirus 2 vaccines with different platforms have been administered worldwide; however, their effectiveness in critical cases of COVID-19 has remained a concern. In this national cohort study, 24 016 intensive care unit (ICU) coronavirus disease-2019 (COVID-19) admissions were included from January to April 2022. The mortality and length of ICU stay were compared between the vaccinated and unvaccinated patients. A total of 9428 (39.25%) patients were unvaccinated, and 14 588 (60.75%) patients had received at least one dose of the vaccine. Compared with the unvaccinated, the first, second, and third doses of vaccine resulted in 8%, 20%, and 33% lower risk of ICU mortality in the adjusted model, with risk ratio (RR): 0.92, 95% confidence interval (CI): 0.84-1.001, RR: 0.80, 95% CI: 0.77-0.83, and RR: 0.67, 95% CI: 0.64-0.71, respectively. The mean survival time was significantly shorter in the unvaccinated versus the fully vaccinated patients (hazard ratio [HR]: 0.84, 95% CI: 0.80-0.88); p < 0.001). All vaccine platforms successfully decreased the hazard of ICU death compared with the unvaccinated group. The duration of ICU stay was significantly shorter in the fully vaccinated than in unvaccinated group (MD, -0.62, 95% CI: -0.82 to -0.42; p < 0.001). Since COVID-19 vaccination in all doses and platforms has been able to reduce the risk of mortality and length of ICU-stay, universal vaccination is recommended based on vaccine availability.

Inflammatory Serum Biomarker Pattern in Emphysema and Chronic Bronchitis Phenotypes of Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Background: COPD exacerbation is characterized by both airway and systemic inflammation. The present study aimed to investigate the relationship between serum levels of some inflammatory biomarkers and the phenotypes of COPD exacerbation. Materials and Methods: This study includes known COPD patients, presenting to a hospital with acute exacerbation of COPD. Serum levels of CRP, ESR, CBC, TNF-α, IL-8, and IL-6 were measured at the time of admission. According to the previously done HRCT, the patients were divided into two groups including emphysema and chronic bronchitis. Levels of serum biomarkers were compared in the two groups. The relationships between biomarkers and duration of hospitalization were assessed too. Results: Comparison of quantitative CRP levels, WBC, and platelet counts did not show a statistically significant difference between emphysema and chronic bronchitis but it was significantly higher than control subjects. Although not statistically significant, ESR level was higher in emphysema. TNF-alpha was 6.0±1.5 ng / ml and 1.5 ng / ml in the emphysema and chronic bronchitis groups, respectively. TNF-α had no significant difference compared to the groups. Although higher than the control group, IL-6 and IL-8 did not show significant differences between emphysema and chronic bronchitis. The two groups did not statistically differ in terms of hospital stay but patients with higher serum TNF-α tended to have longer hospitalization and ICU admission. Conclusion: The present study showed predictably higher inflammatory biomarkers in COPD exacerbation but no significant difference between the two phenotypes of COPD and these two entities could not be discriminated based on inflammatory bio-factors.

Effect of Sofosbuvir on Length of Hospital Stay in Moderate COVID-19 Cases; a Randomized Controlled Trial.

Introduction: Efforts to control the COVID-19 pandemic are still on. This study aimed to evaluate the effect of sofosbuvir on length of hospital stay and complications in COVID-19 cases with moderate severity. Methods: This randomized clinical trial was done on moderate COVID-19 cases, who were admitted to Shohadaye Tajrish Hospital, Tehran, Iran, from 4/2021 to 9/2021. Eligible patients were randomly allocated into two groups of intervention (sofosbuvir) and control, and their outcomes were compared regarding the length of hospital stay and complications. Results: 100 COVID-19 cases were randomly divided into two groups of 50 patients, as the intervention and control groups. The mean age of patients was 50.56 ± 12.23 and 57.1±14.1 years in the intervention and control groups, respectively (p = 0.02). The two groups were similar regarding distribution of gender (p = 0.15), underlying diseases (p = 0.08), the severity of COVID-19 (p = 0.80) at the time of admission, signs and symptoms (p > 0.05), and essential laboratory profile (p > 0.05). The length of hospital stay in the control and intervention groups was 7.7 ± 4.09 days and 4.7±1.6 days, respectively (p = 0.02). None of our patients needed ICU or mechanical ventilation. Conclusion: Sofosbuvir may decrease the length of hospital stay of COVID-19 cases with moderate severity, without a significant effect on the rate of intensive care unit (ICU) need and mortality.

Whole-organ decellularization of the human uterus and in vivo application of the bio-scaffolds in animal models.

PURPOSE: The aim of this investigation was to design a perfusion-based decellularization protocol to provide whole human uterine bio-scaffolds with preserved structural and componential characteristics and to investigate the in vivo properties of the decellularized tissues. METHODS: Eight human uteri, donated by brain-dead patients, were decellularized by perfusion of sodium dodecyl sulfate (SDS) through the uterine arteries using a peristaltic pump. The bio-scaffolds were evaluated and compared with native human uterus regarding histological, immunohistochemical, structural, and bio-mechanical properties, in addition to CT angiographies to examine the preservation of the vascular networks. Subsequently, we obtained acellular patches and implanted them on uterine defects of female Wistar rats to investigate the bio-compatibility and regenerative potential of the bio-scaffolds. Finally, we performed immunostaining to investigate the potential role of circulating stem cells in recellularization of the implanted bio-scaffolds. RESULTS: The outcomes of this investigation confirmed the efficacy of the proposed protocol to provide whole human uterine scaffolds with characteristics and extra-cellular matrix components similar to the native human uterus. Subsequent in vivo studies demonstrated the bio-compatibility and the regenerative potential of the scaffolds and suggested a signaling pathway as an underlying mechanism for the regenerative process. CONCLUSIONS: To the best of our knowledge, this investigation provides the first efficient perfusion-based decellularization protocol for the human uterus to obtain whole-organ scaffolds. The outcomes of this investigation could be employed in future human uterus tissue engineering studies which could ultimately result in the development of novel treatments for female infertile patients.

Organ Procurement From Donors After Brain Death During the COVID-19 Pandemic: Experience of a High Prevalence Country.

OBJECTIVES: Our aim was to compare the donation process before and after the COVID-19 pandemic in an organ procurement unit in Iran and to discuss different strategies to address the impact of the pandemic on the donation process. MATERIALS AND METHODS: All activities including donor detection, donor selection, family approach, donors characteristics, rate of organs per donor, and types of organs were compared between 2 intervals over 18 months (March 2020 to June 2021 [during the COVID19 pandemic] vs November 2018 to February 2020 [before the pandemic]). RESULTS: Before and during the COVID-19 pandemic, there were 218 and 137 brain dead donors with mean age of 42.6 ± 14.5 and 42.8 ± 15.5 years, respectively (P = .82). The prevalence of tumors leading to brain death decreased by more than half during the COVID-19 pandemic (P = .04). There was a 52% increase in cardiac death before organ retrieval during the COVID-19 pandemic, reaching 38% from 25% before COVID-19. During the 2 intervals, the number of organs per donor was 2.3 ± 1.2 and 2.2 ± 1.2 (P = .52). The rate of actual to potential donors before and during the pandemic was significantly different, with 42.16 ± 7.8% before and 29.9 ± 4.8% during the pandemic, mostly as a result of unsuitable donors. The time to obtain family consent during the COVID-19 pandemic was 35.1 ± 8.5 hours, which was a significantly longer length of time than before the pandemic (21.3 ± 12.3 hours; P = .008). CONCLUSIONS: In our organ procurement unit, which encompasses a population of 5.5 million in Tehran, Iran, the number of actual donors was reduced dramatically during the studied pandemic period. However, despite a high workload, all transplant centers and organ procurement units in Iran worked to identify and transplant the available organs to reduce wait list mortality.

Detection of the residual concentration of sodium dodecyl sulfate in the decellularized whole rabbit kidney extracellular matrix.

To optimize rabbit kidney decellularization protocol, using sodium dodecyl sulfate (SDS) as a commonly used detergent, a methylene blue based assay was employed for detecting the minimum nontoxic SDS level for future cell seeding. The rabbit kidney tissues were decellularized with the perfusion-based method and underwent several investigations to determine the efficacy of decellularization in preserving the extracellular matrix (ECM) and cell removal. SDS detection was performed by incubating with methylene blue and subsequent extraction with chloroform. MTT (3-(4, 5-dimethylthiazol-2-yr)-2,5-diphenyltetrazolium bromide) assay and SDS release were also evaluated during the entire process. After the first washing cycle, SDS concentration was 0.036, in 500 mL of the washing liquid, which slowly decreased and reached to 0.009 % after at the end of seventh washing cycle. In the 9th cycle, SDS was gradually decreased and reached to 0.003 %. SDS was significantly released after one week of incubation which ceased after ten washing cycles. The results of MTT assay demonstrated that different cells exhibited various sensitivity levels when exposed to serial concentrations of SDS. Human embryonic kidney cells (HEK293) with 0.003 % threshold for cellular toxicity and 87.4 % cell viability were more resistant compared with mesenchymal stem cells with 0.001 % threshold and 85.4 % cell viability. Colorimetric assay with methylene blue is a straightforward and non-invasive method to detect residual SDS present in tissue and can also prevent ECM destruction after several washings for detergent removal from decellularized tissues.

Serosurvey and Molecular Detection of Toxoplasma gondii in Dogs in Rural Areas of Kazeroun District, Fars Province, Southern Iran.

Background. Toxoplasma gondii is an intracellular protozoan parasite responsible for systemic disease in a wide range of warm-blooded animals. The current study is aimed at evaluating the prevalence of Toxoplasma infection in dogs, using serological and molecular methods in rural areas in Kazeroun Township, Fars province, southern Iran. Methods. Blood samples were obtained from 60 clinically healthy dogs with an age range of 1 to 7 years in three rural areas of Fars province, southern Iran. Sera and buffy coats were used to assess the T. gondii infection using both modified agglutination test (MAT) and real-time PCR. Results. Antibodies against T. gondii were detected in 5 out of 60 (8.3%) dogs by the MAT method, and T. gondii DNA was detected in 17 out of 60 (28.3%) studied animals. There was no significant association between sex and seropositivity to Toxoplasma (p > 0.05). Fair agreement (kappa = 0.27) was seen between molecular and serological findings where three dogs with positive serological results had a positive molecular test. Conclusion. Findings of the present study show a relatively high prevalence of T. gondii infection in dogs in rural areas in Fars province, southern Iran. Finding the parasite genotype in dogs deserves further study.

Application of iPSCs derived pancreatic ß-like cells using pancreatic bio-scaffold.

This study aimed toengineer a pancreatic tissue. Intact rat pancreases were successfully decellularized, and were reseeded with human-induced pluripotent stem cells using different 2D and 3D culture growth factors. The differentiation process was assessed for the presence of a pancreas-like tissue. The histology and SEM analysis revealed cell attachment in all samples, except for the Exp4, and the Flow-cytometry provided 87% viability for the differentiated cells. In Exp1, PDX1 with the positive expression of 2.87±0.06 was dramatically higher than Exp2 with a 2.44±0.06 reaction. NGN3-reactions were 8±0.1 and 6.6±0.2 in Exp1 and Exp2 at P < 0.05, respectively. C-peptide with the expression of 7.5±0.7 in Exp3 was almost equal to that in Exp1 and Exp2. Glucagon (5.1±1) and PDX1 (3.2±0.82) in Exp3 indicated no significant difference. The significant upregulations of pancreatic endocrine markers (PDX1 and NGN3), and the cell-specific glucose transporter (GLUT2) were observed in the differentiated IPCs in the 3D culture of Exp2 after 21 days. The highest insulin and C-peptide concentrations were observed in Exp2. In Exp3, insulin secretion in response to high glucose and 10 mM arginine was 42.43 ±6.34 µU/ml. A decellularized pancreas in the presence of hiPSCs and growth factors could be efficiently used as a natural scaffold.

Molecular and serological evaluation of zoonotic visceral leishmaniasis in dogs in a rural area of Fars province, southern Iran, as a source of Leishmania infantum infection.

Canine visceral leishmaniasis (CVL) is endemic in the southern parts of Iran. The current study aimed at molecular and serological evaluation of zoonotic visceral leishmaniasis in dogs in Fars province, southern Iran. Blood samples were collected from 60 dogs in the three villages in Fars Province. Serum samples were tested for antibodies against L. infantum by direct agglutination test (DAT). DNA was extracted from each dog's buffy coat and tested by PCR, targeting the Leishmania ITS-2 region. From a total of 60 studied dogs, 25 (41.7%) were female, and 35 (58.3%) were male. Dogs' age ranged from 1 to 7 years, with a mean age of 2.97 (±1.4) years. Anti-Leishmania antibodies were detected in sera samples of 28 (46.7%) dogs, (titre ≥ 1:320). Out of 28 seropositive cases, 13 (46.4%) were female, and 15 (53.6%) were male. Association between seropositivity and dogs' clinical signs was statistically significant (p < .05). Leishmania DNA was detected in the buffy coat of 3 of 60 studied dogs which were all seropositive by DAT. The PCR products were sequenced and molecular analysis showed that two of the isolates were Leishmania infantum, and one was L. tropica. The high proportion of seropositive dogs indicates the important role of these animals in the epidemiology of the disease in the region. Infected dogs with or without signs, especially those that are molecularly positive, can act as an active reservoir of the disease in the area.

Molecular and Serological Evaluation of Neospora caninum Infection in Dogs from a Rural Setting in Fars Province, Southern Iran.

BACKGROUND: Dogs, as the definitive host of Neospora caninum, are important in the epidemiology of this parasitic infection. We aimed to determine the prevalence of N. caninum infection in a dog population from a rural setting in Fars Province, Southern Iran, using a combination of molecular and serological techniques. METHODS: This cross-sectional study was carried out in Nov 2018 in three rural districts, Sar Mashhad, HosseinAbad, and Tolesaman located in Kazeroun Township in Fars province, southern Iran. Blood samples were taken from 60 stray and household dogs. Dogs' sera were tested for antibodies against N. caninum, using a Neospora-Modified Agglutination Test. Moreover, dogs' buffy coats were tested for Neospora DNA, using a molecular method. RESULTS: Anti-Neospora antibodies were detected in sera of 4 out of 60 dogs, corresponding to a seroprevalence rate of 6.7%. Out of 25 female dogs, 1 was seropositive and of 35 males, 3 were seropositive, yet the differences were not statistically significant. The infection was more prevalent in adult dogs (> 12 months), nevertheless, the differences between age and Neospora seropositivity was not statistically significant. N. caninum DNA was not detected in the buffy coat of any of the studied dogs. CONCLUSION: Findings of the study indicate that N. caninum is a common infection in dogs in rural areas of Fars province in southern Iran. The infected dogs might be a potentially important source of N. caninum infection to livestock in the area.