Long COVID in Immunocompromised and Immunocompetent Patients: A Clinical, Morphologic, and Virologic Portrait.

CONTEXT.­: Coronavirus disease 2019 (COVID) primarily affects the lung and can lead to chronic/post-COVID syndrome. Some insights about late pulmonary changes occurring in patients recovering from COVID have been published, but the evidence of detailed pathologic changes coming from follow-up care patients with long COVID is limited. OBJECTIVE.­: To evaluate tissue morphologic and viral features in transbronchial biopsies of long COVID patients (immunocompetent and immunocompromised). DESIGN.­: This retrospective observational study included 18 patients (9 immunocompetent and 9 immunocompromised) who were consecutively referred to outpatient clinic for post-COVID pneumonia, undergoing transbronchial biopsy. Several histologic changes were analyzed by computer-assisted morphometric analysis. As organizing pneumonia (OP) was consistently detected, fibrosis and inflammation were also evaluated in transbronchial biopsies from 28 control patients with histologic confirmation of OP. Tissue SARS-CoV-2 and the subgenomic transcripts were investigated. Morphologic findings were correlated with clinical and radiologic data. RESULTS.­: Long COVID patients showed lower inflammation than controls (P < .001) despite a similar fibrotic extension. When considering separately the 2 long COVID groups, the same inflammatory infiltrate extension was found, whereas a higher fibrotic remodeling characterized the immunocompetent subgroup (P = .05). Molecular investigation showed that SARS-CoV-2 was present in tissue samples obtained from 3 long COVID patients. CONCLUSIONS.­: Long COVID patients showed a peculiar OP pattern, with more vascular and fibrotic changes. SARS-CoV-2 RNA, even in replicative status, can be detected in lung biopsies of both immunocompetent and immunocompromised patients. This pilot study is a forerunner of more in-depth lung tissue investigations to gain a better understanding of long COVID pathobiology.

Blood Eosinophils Matter in Post-COVID-19 Pneumonia.

BACKGROUND: Even after the development of vaccines, SARS-CoV-2 continues to cause severe pneumonia all over the world. Consequently, in order to improve the management of patients and optimize the use of resources, predictors of disease severity and lung complications after COVID-19 pneumonia are urgently needed. Blood cell count is an easily available and reproducible biomarker. With this study, we aimed to explore the role of eosinophils in predicting disease behavior and pulmonary sequelae at first follow-up with computed tomography (CT). METHODS: we evaluated blood cell count and other inflammatory markers, both at baseline and during hospitalization, in a large population of hospitalized COVID-19 patients. RESULTS: 327 patients were finally enrolled, 214 were classified as low-intensity medical care (LIMC) and 113 as high-intensity medical care. Eosinophils were higher at discharge in the HIMC group [0.1 (0-0.72) vs. 0.05 (0-0.34) × 109/L; p < 0.0001]. Moreover, in the multivariable analysis, age ≥ 62 years (OR 1.76 (1.05-2.8) p = 0.03) and Δ eosinophils ≥ 0.05 (OR 1.75 (1.05-2.9) p = 0.03) were two independent predictors of residual lung abnormalities in the whole patient population at first follow-up. CONCLUSIONS: an eosinophil increase during hospitalization could be a potential predictor of pulmonary sequelae in surviving patients after COVID-19 pneumonia.

Bone marrow edema in children: chronic nonbacterial osteomyelitis and its mimickers.

Bone marrow is a highly cellular tissue undergoing significant developmental and physiologic changes with age. Indeed, with maturation from pediatric to the adult age there is a progressive, centrifugal conversion from red to yellow bone marrow. Histological characteristics of bone marrow are reflected in MR image signal. MR is therefore extremely sensitive in detecting pathological changes which are mostly characterized by increased free water causing high signal intensity on T2. Among the numerous diseases causing bone marrow edema in children chronic nonbacterial osteomyelitis (CNO) certainly has to be mentioned. This idiopathic inflammatory disorder is characterized by nonspecific migrating symptoms like skeletal pain with phases of exacerbations and relapses with alternating acute and chronic MR signs and it is often a diagnosis of exclusion. Hence, with bone marrow edema, various features at imaging should be considered to differentiate malignancies such as osseous lymphoma, osteosarcoma, and Ewing's sarcoma as well as benign lesions like osteomyelitis, post-traumatic, or post-treatment bone marrow edema. The aim of this review is to recall the main characteristics of CNO and provide an overview of its main mimickers highlighting similarities and differences.

Clinical information on imaging referrals for suspected or known axial spondyloarthritis: recommendations from the Assessment of Spondyloarthritis International Society (ASAS).

OBJECTIVES: This study aims to establish expert consensus recommendations for clinical information on imaging requests in suspected/known axial spondyloarthritis (axSpA), focusing on enhancing diagnostic clarity and patient care through guidelines. MATERIALS AND METHODS: A specialised task force was formed, comprising 7 radiologists, 11 rheumatologists from the Assessment of Spondyloarthritis International Society (ASAS) and a patient representative. Using the Delphi method, two rounds of surveys were conducted among ASAS members. These surveys aimed to identify critical elements for imaging referrals and to refine these elements for practical application. The task force deliberated on the survey outcomes and proposed a set of recommendations, which were then presented to the ASAS community for a decisive vote. RESULTS: The collaborative effort resulted in a set of six detailed recommendations for clinicians involved in requesting imaging for patients with suspected or known axSpA. These recommendations cover crucial areas, including clinical features indicative of axSpA, clinical features, mechanical factors, past imaging data, potential contraindications for specific imaging modalities or contrast media and detailed reasons for the examination, including differential diagnoses. Garnering support from 73% of voting ASAS members, these recommendations represent a consensus on optimising imaging request protocols in axSpA. CONCLUSION: The ASAS recommendations offer comprehensive guidance for rheumatologists in requesting imaging for axSpA, aiming to standardise requesting practices. By improving the precision and relevance of imaging requests, these guidelines should enhance the clinical impact of radiology reports, facilitate accurate diagnosis and consequently improve the management of patients with axSpA.

Targeting senescence induced by age or chemotherapy with a polyphenol-rich natural extract improves longevity and healthspan in mice.

Accumulating senescent cells within tissues contribute to the progression of aging and age-related diseases. Botanical extracts, rich in phytoconstituents, present a useful resource for discovering therapies that could target senescence and thus improve healthspan. Here, we show that daily oral administration of a standardized extract of Salvia haenkei (Haenkenium (HK)) extended lifespan and healthspan of naturally aged mice. HK treatment inhibited age-induced inflammation, fibrosis and senescence markers across several tissues, as well as increased muscle strength and fur thickness compared with age-matched controls. We also found that HK treatment reduced acutely induced senescence by the chemotherapeutic agent doxorubicin, using p16LUC reporter mice. We profiled the constituent components of HK by mass spectrometry, and identified luteolin-the most concentrated flavonoid in HK-as a senomorphic compound. Mechanistically, by performing surface plasmon resonance and in situ proximity ligation assay, we found that luteolin disrupted the p16-CDK6 interaction. This work demonstrates that administration of HK promotes longevity in mice, possibly by modulating cellular senescence and by disrupting the p16-CDK6 interaction.

Standardized reporting of spine and sacroiliac joints in axial spondyloarthritis MRI: from the ESSR-Arthritis Subcommittee.

OBJECTIVES: Apply a modified Delphi-based approach and produce a practical, radiology-specific set of definitions for interpretation and standardization of the multiple MRI findings in axial spondyloarthritis (ax-SpA), specifically to aid the general radiologist with a musculoskeletal interest, working with gold standard basic MRI protocols. MATERIALS AND METHODS: We report the results of a modified Delphi-based consensus of 35 experts from 13 countries in the Arthritis Subcommittee of the European Society of Musculoskeletal Radiology (ESSR). Seventeen definitions were created (i.e., nine for the spine and eight for the sacroiliac joint) and two Delphi rounds were conducted on an electronic database, collated and revised by the project leader with agreement. Group leads were appointed for each definition following the first round. Final definitions included only those that reached a consensus > 80%; if > 50% agreed on exclusion consensus, definitions were excluded. Final results have been shared during the Arthritis meeting at the Annual ESSR Congress. RESULTS: Fourteen definitions, eight for the spine and six for the sacroiliac joint were agreed for standardized reporting. Andersson's, anterior corner sclerotic and costovertebral joint inflammatory lesions of the spine, with active and non-active erosions, and fat metaplasia of the sacroiliac joint reaching the highest consensus (≥ 95%). More than 50% of the experts agreed to exclude joint space inflammation in the sacroiliac joint and tissue backfill. Syndesmophytes reached 76% agreement. CONCLUSIONS: Agreed definitions by expert radiologists using a modified Delphi process, should allow standardized actionable radiology reports and clarity in reporting terminology of ax-SpA. CLINICAL RELEVANCE STATEMENT: The proposed definitions will support reporting from musculoskeletal and general radiologists working with gold-standard basic MRI, improve confidence in lesion assessment, and standardize terminology to provide actionable reports on MRI in patients with ax-SpA. KEY POINTS: Experts applied a modified Delphi method to optimize the definitions of MRI findings of ax-SpA. After two Delphi rounds and one in-person meeting, fourteen definitions reached the agreement threshold. These consensus-based definitions will aid in actionable reporting specifically for the general radiologist with a musculoskeletal interest.

Update on Rheumatic Diseases in Clinical Practice: Recent Concepts and Developments.

Diagnostic imaging is essential in the diagnostic process of rheumatic diseases. Given the heterogeneity of this group of diseases and the tremendous impact of novel therapeutic options, guidelines and recommendations regarding the optimal choice of the most appropriate technique/s are continuously revised and radiologists should always be up-to-date. Last, because of the continuous technological innovations, we will assist to the progressive application of advanced techniques and tools in rheumatology.

Whole-Body Magnetic Resonance Imaging in Rheumatology.

This review focuses on the most frequent whole-body MRI applications in patients with rheumatological pathologies, for which this tool can be helpful to both radiologists and clinicians. It reports technical aspects of the acquisition of both 1.5 and 3.0 T scanners. The article lists the main findings that help radiologists during the evaluation of a specific pathology, both in the diagnostic phase and during follow-up.

Automatic assessment of body composition in children with lymphoma: results of a [18F]FDG-PET/MR study.

OBJECTIVES: To use Dixon-MR images extracted from [18F]FDG-PET/MR scans to perform an automatic, volumetric segmentation and quantification of body composition in pediatric patients with lymphoma. MATERIALS AND METHODS: Pediatric patients with lymphoma examined by [18F]FDG-PET/MR at diagnosis and restaging were included. At each time point, axial fat and water Dixon T1w images of the thighs were automatically segmented and muscle volume, subcutaneous, intramuscular, and intermuscular fat volume were quantified. The metabolic activity of the largest nodal lesion and of muscles and subcutaneous fat was recorded. The paired samples t-test and Spearman's correlation coefficient were applied to evaluate potential differences between the two time points and the relationship between metabolic and body composition metrics, respectively. By logistic regression analysis, the prognostic role of the investigated variables was assessed. The applied significance level was p < 0.05 for all analyses. RESULTS: Thirty-seven patients (mean age ± SD 14 ± 3-years-old; 20 females) matched the inclusion criteria. After chemotherapy (interval between the two PET/MR scans, 56-80 days; median 65 days), muscle volume significantly decreased (629 ± 259 cm3 vs 567 ± 243 cm3, p < 0.001) while subcutaneous, intramuscular and intermuscular fat increased (476 ± 255 cm3 vs 607 ± 254 cm3, p < 0.001; 63 ± 20 cm3 vs 76 ± 26 cm3, p < 0.001; 58 ± 19 cm3 vs 71 ± 23 cm3, p < 0.001); the metabolic activity of the main nodal lesion, muscles, and subcutaneous fat significantly decreased (p < 0.05, each). None of the examined variables acted as predictors of the response to treatment (p = 0.283). A strong correlation between BMI and subcutaneous fat volume at diagnosis (r = 0.675, p < 0.001) and restaging (r = 0.600, p < 0.001) emerged. CONCLUSIONS: The proposed method demonstrated that pediatric patients with lymphoma undergo muscle loss and an increase of subcutaneous fat during treatment. CLINICAL RELEVANCE STATEMENT: The proposed automatic and volumetric MR-based assessment of body composition in children with lymphoma can be used to monitor the effect of chemotherapy and may guide tailored exercise programs during chemotherapy. KEY POINTS: T1w Dixon images can be used for the automatic segmentation and quantification of body composition. Muscle and subcutaneous fat volume do not act as predictors of the response to treatment in children with lymphoma. Chemotherapy induces changes in body composition in children with lymphoma.

The role of pathologists in the diagnosis of occupational lung diseases: an expert opinion of the European Society of Pathology Pulmonary Pathology Working Group.

Occupational lung/thoracic diseases are a major global public health issue. They comprise a diverse spectrum of health conditions with complex pathology, most of which arise following chronic heavy workplace exposures to various mineral dusts, metal fumes, or following inhaled organic particulate reactions. Many occupational lung diseases could become irreversible; thus accurate diagnosis is mandatory to minimize dust exposure and consequently reduce damage to the respiratory system. Lung biopsy is usually required when exposure history is inconsistent with imaging, in case of unusual or new exposures, in case of unexpected malignancy, and in cases in which there are claims for personal injury and legal compensation. In this paper, we provide an overview of the most frequent occupational lung diseases with a focus on pathological diagnosis. This is a paper that summarizes the expert opinion from a group of European pathologists, together with contributions from other specialists who are crucial for the diagnosis and management of these diseases. Indeed, tight collaboration of all specialists involved in the workup is mandatory as many occupational lung diseases are misdiagnosed or go unrecognized. This document provides a guide for pathologists in practice to facilitate the accurate diagnosis of occupational lung disease. The review article reports relevant topics discussed during an educational course held by expert pathologists, active members of the Pulmonary Pathology Working Group of the European Society of Pathology. The course was endorsed by the University of Padova as a "winter school" (selected project in the call for "Shaping a World-class University" 2022).

Neonatal cerebral ultrasound: anatomical variants and age-related diseases.

Cerebral ultrasound is a non-invasive imaging technique widely used for the assessment of brain anatomy and diseases in neonates and infants. Indeed, it allows a precise characterization of common variants such as cavum septum pellucidum or diseases like intraventricular hemorrhage. The aim of this pictorial review is to provide a comprehensive overview of the main ultrasound features of the most common cerebral anatomical variants and disorders detectable by cerebral ultrasound using an age-related approach which could support non-subspecialized radiologists.

Reporting Sacroiliac Joint Imaging Performed for Known or Suspected Axial Spondyloarthritis: Assessment of SpondyloArthritis International Society Recommendations.

Whereas previous projects attempted to standardize imaging in patients with axial spondyloarthritis (axSpA), few studies have been published about the need for specific details regarding the image acquisition and lesions that may be less familiar to general radiologists. This work reports consensus recommendations developed by the Assessment of SpondyloArthritis International Society (ASAS) that aim to standardize the imaging reports in patients suspected of having or with known axSpA. A task force consisting of radiologists and rheumatologists from ASAS and one patient representative formulated two surveys that were completed by ASAS members. The results of these surveys led to the development of 10 recommendations that were endorsed by 73% (43 of 59) of ASAS members. The recommendations are targeted to the radiologist and include best practices for the inclusion of clinical information, technical details, image quality, and imaging findings in radiology reports. These recommendations also emphasize that imaging findings that indicate differential diagnoses and referral suggestions should be included in the concluding section of the radiology report. With these recommendations, ASAS aims to improve the diagnostic process and care for patients suspected of having or with known axSpA.

Systemic sclerosis sine scleroderma is more aggressive in children than in adults.

OBJECTIVES: To compare the clinical and laboratory features of paediatric SSc sine scleroderma (ssJSSc) with adult-onset ssSSc. METHODS: Demographic, clinical and laboratory data of ssJSSc, retrospectively retrieved from our hospital medical records, case reports from the literature and from the Pediatric Rheumatology European Society JSSc registry, were compared with the Padua cohort of adult patients with ssSSc. Patients were defined as having ssSSc if they never had skin involvement but all the following features: (i) RP and/or digital vasculopathy, (ii) positive ANA, (iii) internal organs involvement typical of scleroderma and (iv) no other defined CTD. RESULTS: Eighteen juvenile and 38 adult-onset ssSSc patients, mean disease duration 5.8 and 9.7 years, respectively, entered the study. The frequency of females affected was significantly lower in ssJSSc (38.9% vs 89.5%, P < 0.0001). When compared with adults, ssJSSc displayed fewer SSc-specific capillaroscopy abnormalities (68.8% vs 94.7%, P = 0.02) while having significantly higher vascular (digital pitting scars, ulcers 35.3% vs 10.5%, P = 0.042), respiratory (50.0% vs 23.7%, P = 0.02) and cardiac (50.0% vs 2.6%, P < 0.0001) involvement. The outcome was significantly worse in ssJSSc as six patients (33%) died (n = 3) or reached an end-stage organ failure (n = 3) in comparison with only two deaths (5.3%) in the adult cohort. ACA were significantly lower in children (20.0% vs 68.4%, P = 0.001) while no difference was noted for other SSc-specific autoantibodies. CONCLUSION: Compared with adults where ssSSc generally has an indolent course, children present with aggressive disease that heralds a worse prognosis characterized by high cardiorespiratory morbidity and mortality.

Advanced Imaging of Gout and Other Inflammatory Diseases Around the Knee.

The knee is one of the most commonly affected joints in the course of inflammatory arthropathies, such as crystal-induced and autoimmune inflammatory arthritis. The latter group includes systemic connective tissue diseases and spondyloarthropathies. The different pathogenesis of these entities results in their varied radiologic images. Some lead quickly to joint destruction, others only after many years, and in the remaining, destruction will not be a distinguishing radiologic feature.Radiography, ultrasonography, and magnetic resonance imaging have traditionally been the primary modalities in the diagnosis of noninflammatory and inflammatory arthropathies. In the case of crystallopathies, dual-energy computed tomography has been introduced. Hybrid techniques also offer new diagnostic opportunities. In this article, we discuss the pathologic findings and imaging correlations for crystallopathies and inflammatory diseases of the knee, with an emphasis on recent advances in their imaging diagnosis.

Endobronchial solitary fibrous tumors: An enigma for diagnosis.

Solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms constituting less than 2% of all soft tissue tumors. They typically originate in the thoracic cavity, mainly in the pleura, but can also occur in other various sites such as lung parenchyma, pericardium, and bronchus. In this study, a 49-year-old non-smoking female with a history of allergies presented to our pulmonary clinic with a chronic cough. An explorative bronchoscopy revealed an intrabronchial mass in the left superior bronchi, and a 68 Ga-DOTATOC positron emission computed tomography suggested a carcinoid tumor. Subsequent pulmonary segmentectomy unveiled a well-circumscribed polypoid lesion diagnosed as a low-grade bronchus SFT through histopathological and immunohistochemical assessments. The patient was asymptomatic after surgical excision and showed no other lesion during the 6-month follow-up. The endobronchial location of SFT is uncommon, with only a few reported cases in the literature, underscoring the necessity of considering various differential diagnoses, including carcinoid, mucoepidermoid carcinoma, endobronchial pleomorphic adenoma, hamartoma, leiomyoma, and metastasis, depending on location and imaging features. This report underscores the importance of careful histological and immunohistochemical evaluation in understanding and appropriately stratifying the risk associated with polypoid lesions.

Severe starvation and restraint in a 47-year-old woman: Clinical, autopsy and histopathological evidence of abuse and neglect.

The recognition of abuse and/or neglect still represents a challenge for both clinicians and forensic pathologists. Whereas abusive behaviors have been largely described among pediatric and elderly populations, adults' abuse and neglect is less frequently encountered, and therefore investigated. We report a case of a middle-aged woman without any known organic or psychiatric disorders who died of a multiple organ failure (MOF) due to extreme cachexia and bed-resting syndrome. The integration of all clinical, autopsy and histopathological data highlighted a picture of severe malnutrition, restraint, and widespread traumatic injuries related to abuse and neglect. We believe that the case here presented could be useful for both clinicians and forensic pathologists as it underlines once again the importance of collecting and integrating all medical evidence (both in the ante- and post-mortem settings) for reconstructing the most probable pathophysiology of disorders and injuries, comparing that reconstruction with the allegations of the caregivers, and thus identifying any potential abuse and/or neglect behaviors.

The Precision, Inter-Rater Reliability, and Accuracy of a Handheld Scanner Equipped with a Light Detection and Ranging Sensor in Measuring Parts of the Body-A Preliminary Validation Study.

BACKGROUND: Anthropometric measurements play a crucial role in medico-legal practices. Actually, several scanning technologies are employed in post-mortem investigations for forensic anthropological measurements. This study aims to evaluate the precision, inter-rater reliability, and accuracy of a handheld scanner in measuring various body parts. METHODS: Three independent raters measured seven longitudinal distances using an iPad Pro equipped with a LiDAR sensor and specific software. These measurements were statistically compared to manual measurements conducted by an operator using a laser level and a meterstick (considered the gold standard). RESULTS: The Friedman test revealed minimal intra-rater variability in digital measurements. Inter-rater variability analysis yielded an ICC = 1, signifying high agreement among the three independent raters. Additionally, the accuracy of digital measurements displayed errors below 1.5%. CONCLUSIONS: Preliminary findings demonstrate that the pairing of LiDAR technology with the Polycam app (ver. 3.2.11) and subsequent digital measurements with the MeshLab software (ver. 2022.02) exhibits high precision, inter-rater agreement, and accuracy. Handheld scanners show potential in forensic anthropology due to their simplicity, affordability, and portability. However, further validation studies under real-world conditions are essential to establish the reliability and effectiveness of handheld scanners in medico-legal settings.

Lung relapse pattern in children with metastatic Wilms tumor.

Wilms tumor is the most common pediatric renal cancer, and lungs represent the major site of metastasis and recurrence. Relapse occurs in 15%, months or years after treatment; so due to the small sample, acquiring more data about the pattern of lung relapse remains a challenge. The aim of our study was to evaluate if pulmonary relapse, detected by computed tomography (CT), occurred at the initial site of lung metastases or in a different location. According to our data, the CT pattern of lung relapse showed high probability of recurrence at the same site of initial metastasis.

An unusual case of corpse concealment driven by emotional distress.

Corpse concealment involves hiding a body for criminal purposes for many different reasons, such as destroy evidence of a murder or avoid the discovery of the victim. Although defendants could argue that they did not conceal the corpse with any criminal intent, but rather to spare themselves or others from emotional distress or to honor the wishes of the deceased. However, these arguments are often challenging to substantiate, and defendants may encounter significant legal obstacles when attempting to justify their actions. Herein, we report a case involving the concealment of a woman's corpse by her father. Autopsy and histological investigations were significantly limited due to the advanced decomposition of the body. Nevertheless, by integrating these data with radiological findings obtained from total body CT and micro-CT of the larynx-hyoid complex, hanging was deemed the cause of death. Additionally, the psychological evaluation of the father indicated that the act of concealment was motivated by emotions rather than criminal intent.

Detection of butterfly fractures of long bones through multi-slice computed tomography and micro-computed tomography.

Motor-vehicle accidents often result in lower limb injuries with biosseous fractures. The present study aimed at comparing multi-slice computed tomography (MS-CT), micro-computed tomography (micro-CT) and external fractography for the analyses of experimentally produced biosseus leg fractures. Briefly, 48 human legs amputated for medical reasons were defleshed and then experimentally fractured using a 3-point dynamic bending model (70,6 J of impact energy at the middle of the anterior surface of the tibia) producing 38 biosseous and 10 mono-osseous fractures with a total of 86 fractured bones. External fractography detected 63 (73,2%) "butterfly" fractures (24 (27,9%) complete and 39 (45,3%) incomplete), 14 (16,3%) "oblique" fractures, 6 (7,0%) "comminuted" fractures and 3 (3,5%) "transverse" fractures. Forty-three (43) of the 48 included legs displayed at least one butterfly fracture located at the tibia or fibula. MS-CT correctly detected and classified 16 complete and 20 incomplete butterfly fractures, failing to properly classify 27 fractures; 19 of these misclassifications led to an interpretative error on the trauma direction (i.e., 16 incomplete butterfly fractures classified as oblique fractures and 3 incomplete butterfly fractures classified as transverse). Micro-CT correctly detected and classified 22 complete and 37 incomplete butterfly fractures, failing to properly classify 4 fractures; two of these misclassifications led to an interpretative error on the trauma direction (i.e., two incomplete butterfly fractures classified as oblique fractures). Although further studies evaluating a wider number of fractures and fracture patterns are required to drive any definitive conclusions, this preliminary experimental investigation showed that MS-CT and micro-CT represent useful tools for reconstructing the morphology of leg fractures and could be crucial for trauma analysis in the forensic context. MS-CT could be used as a screening tool, micro-CT as second level analysis and external/internal fractography as third level, confirmatory analysis.