BACKGROUND: Extrapulmonary tuberculosis (EPTB) makes for 25% of all instances of tuberculosis (TB) patients. The enigmatic clinical presentation of EPTB makes identification difficult since it simulates other chronic conditions such as neoplastic and inflammatory disorders and could culminate in treatment that is either insufficient or not required. For an affirmative and confirmed diagnosis, a substantial level of suspicion is imperative. The paucibacillary feature of EPTB makes diagnosis extremely difficult and necessitates the use of many diagnostic methods to arrive at a precise diagnosis. In December 2010, the World Health Organization recommended using GeneXpert/cartridge-based nucleic acid amplification test (CBNAAT) for the initial assessment of suspected cases of EPTB. Furthermore, fine-needle aspiration cytology (FNAC), Ziehl-Neelsen (ZN) stain, and the CBNAAT have to be utilized to exclude other possible origins of granulomatous inflammation. The goal of the current investigation is to comprehend how FNAC and ZN stains relate to CBNAAT and their diagnostic value.
METHODS: The evaluation included all suspected instances of tubercular lymphadenopathy, and adequate aspirates were obtained from the site of the enlarged cervical lymph nodes. Smears were made following FNAC and stained with ZN stain as well as hematoxylin and eosin stain. Simultaneously, CBNAAT and culture evaluations were conducted on the same aspirates. This cross-sectional study took place at a tertiary care center and encompassed 200 individuals with clinical manifestations of EPTB.
RESULTS: There were 200 cases of suspected tubercular lymphadenitis (TBLN). According to the FNAC results, TBLN was detected in 71 (47.6%) of these 200 cases, followed by necrotizing lymphadenitis in 56 (37.5%), chronic caseating granulomatous lymphadenitis in 47 (31.5%), and reactive lymphadenitis in 26 (17.4%). They were correlated with CBNAAT results, which showed that all instances of tuberculous lymphadenitis, 85.71% of cases of necrotizing lymphadenitis, 55.32% of cases of chronic caseating granulomatous lymphadenitis, and 2 (7.69%) cases of reactive lymphadenitis were CBNAAT positive.
CONCLUSIONS: CBNAAT should be utilized with FNAC and ZN staining to diagnose EPTB. The CBNAAT assay demonstrated a significant advantage in the identification of previously unidentified FNAC patients. Despite being a simple diagnostic tool, FNAC has a lower specificity and significantly lower precision than CBNAAT in correctly identifying cases of EPTB because it exhibits similar cytomorphological characteristics with lesions that are not associated with TB.

OBJECTIVE: To compare the staining quality between rapid hematoxylin and eosin (H&E) staining and routine H&E staining of frozen breast tissue sections.
METHODS: In this cross-sectional observational study, 120 frozen breast tissue sections were randomly assigned to rapid or routine H&E staining (n = 60 per group). Rapid H&E staining used a 7:1 mixture of modified Gill\'s hematoxylin and alcohol-soluble 1% eosin Y. The staining quality of each section was evaluated and scored. A score of >7 was considered excellent, a score of 6 to 7 good, and a score of ≤5 poor.
RESULTS: The staining time for rapid staining was approximately 3 minutes, whereas that of routine staining was approximately 12 minutes. There were no significant differences in the staining quality scores or proportions of sections in each grade between the two staining methods. The proportions of sections that were classified as excellent or good were 96.7% and 98.3% for rapid and routine staining, respectively.
CONCLUSIONS: In frozen breast tissue sections, rapid H&E staining may provide staining quality that is comparable to that of routine staining, while markedly reducing the staining time.

BACKGROUND: Precision surgery for liver tumors favors laparoscopic anatomical liver resection (LALR), involving the removal of specific liver segments or subsegments. Indocyanine green (ICG)-negative staining is a commonly used method for defining resection boundaries but may be prone to failure. The challenge arises when ICG staining fails, as it cannot be repeated during surgery. In this study, we employed the virtual liver segment projection (VLSP) technology as a salvage approach for precise boundary determination. Our aim was to assess the feasibility of the VLSP to be used for the determination of the boundaries of the liver resection in this situation.
METHODS: Between January 2021 and June 2023, 12 consecutive patients undergoing subsegment-oriented LALR were included in this pilot series. The VLSP technology was utilized to define the resection boundaries at the time of ICG-negative staining failure. Routine surgical parameters and short-term outcomes were evaluated to assess the safety of VLSP in this procedure. In addition, its feasibility was assessed by analyzing the accuracy between the predicted resected liver volume (PRLV) and actual resected liver volume (ARLV).
RESULTS: Of the 12 enrolled patients, the mean operation time was 444.58 ± 101.70 min (range 290-570 min), with a mean blood loss of 125.00 ± 96.53 ml (range 50-400 mL). One patient (8.3%) was converted to laparotomy for subsequent parenchymal transection, four (33.3%) received blood transfusions and four (33.3%) had postoperative complications. All patients received an R0 resection. The Pearson correlation coefficient (r) between PRLV and ARLV was 0.98 (R2 = 0.96, p < 0.05), and the relative error (RE) was 8.62 ± 6.66% in the 12 patients, indicating agreement.
CONCLUSIONS: Failure of intraoperative ICG-negative staining during subsegment-oriented LALR is possible, and VLSP may be an alternative to define the resection boundaries in such cases.

BACKGROUND: Each high-risk HPV genotype has different oncogenic potential, and the risk of CIN3+ varies according to genotype. We evaluated the performance of different strategies of HPV-positivity triage combining cytology, p16/ki67 dual staining (DS), and extended genotyping.
METHODS: Samples from 3180 consecutive women from the NTCC2 study (NCT01837693) positive for HPV DNA at primary screening, were retrospectively analyzed by the BD Onclarity HPV Assay, which allows extended genotyping. Genotypes were divided into three groups based on the risk of CIN3+. HPV DNA-positive women were followed up for 24 months or to clearance.
RESULTS: Combining the three groups of genotypes with cytology or DS results we identify a group of women who need immediate colposcopy (PPV for CIN3+ from 7.8 to 20.1%), a group that can be referred to 1-year HPV retesting (PPV in those HPV-positive at retesting from 2.2 to 3.8), and a group with a very low 24-month CIN3+ risk, i.e. 0.4%, composed by women cytology or DS negative and positive for HPV 56/59/66 or 35/39/68 or negative with the Onclarity test, who can be referred to 3-year retesting.
CONCLUSIONS: Among the baseline HPV DNA positive/cytology or DS negative women, the extended genotyping allows to stratify for risk of CIN3+, and to identify a group of women with a risk of CIN3+ so low in the next 24 months that they could be referred to a new screening round after 3 years.
BACKGROUND: Italian Ministry of Health (grant number RF-2009-1536040). Hologic-Genprobe, Roche Diagnostics, and Becton & Dickinson provided financial and non-financial support.

BACKGROUND: Chondrosarcomas are rare malignant bone tumors diagnosed by analyzing radiological images and histology of tissue biopsies and evaluating features such as matrix calcification, cortical destruction, trabecular penetration, and tumor cell entrapment.
METHODS: We retrospectively analyzed 16 cartilaginous tumor tissue samples from three patients (51-, 54-, and 70-year-old) diagnosed with a dedifferentiated chondrosarcoma at the femur, a moderately differentiated chondrosarcoma in the pelvis, and a predominantly moderately differentiated chondrosarcoma at the scapula, respectively. We combined a hematein-based x-ray staining with high-resolution three-dimensional (3D) microscopic x-ray computed tomography (micro-CT) for nondestructive 3D tumor assessment and tumor margin evaluation.
RESULTS: We detected trabecular entrapment on 3D micro-CT images and followed bone destruction throughout the volume. In addition to staining cell nuclei, hematein-based staining also improved the visualization of the tumor matrix, allowing for the distinction between the tumor and the bone marrow cavity. The hematein-based staining did not interfere with further conventional histology. There was a 5.97 ± 7.17% difference between the relative tumor area measured using micro-CT and histopathology (p = 0.806) (Pearson correlation coefficient r = 0.92, p = 0.009). Signal intensity in the tumor matrix (4.85 ± 2.94) was significantly higher in the stained samples compared to the unstained counterparts (1.92 ± 0.11, p = 0.002).
CONCLUSIONS: Using nondestructive 3D micro-CT, the simultaneous visualization of radiological and histopathological features is feasible.
CONCLUSIONS: 3D micro-CT data supports modern radiological and histopathological investigations of human bone tumor specimens. It has the potential for being an integrative part of clinical preoperative diagnostics.
CONCLUSIONS: • Matrix calcifications are a relevant diagnostic feature of bone tumors. • Micro-CT detects all clinically diagnostic relevant features of x-ray-stained chondrosarcoma. • Micro-CT has the potential to be an integrative part of clinical diagnostics.

This in vitro study aimed to evaluate the feasibility of quantitative light-induced fluorescence (QLF) technology for detecting the presence and severity of microleakage of pit and fissure sealants. The areas of interest (AOIs) were 160 pits and fissures of 40 extracted permanent teeth. Fluorescent images were acquired using a QLF device, and the maximum fluorescence loss ΔFmax of each AOI was analyzed. After staining and cross-sectioning of the teeth, histological dye penetration was scored on a scale of 0 to 3. The relationship between ΔFmax and microleakage depth was analyzed, and the areas under the curve (AUCs) were calculated. The │ΔFmax│ increased as microleakage depth increased. The ΔFmax values of microleakage areas showed a strong significant correlation with the histological scores of dye penetration (r = - 0.72, P = 0.001). AUC analysis showed a high diagnostic accuracy for microleakage depth (AUC = 0.83-0.91). The highest AUC of 0.91 was found when differentiating the outer half microleakage of the sealant (histological score 0 vs. 1-3). QLF technology is effective in assessing the presence and severity of microleakage, suggesting its potential for noninvasive detection and monitoring of sealant microleakage in clinical settings.

OBJECTIVE: To study the correlation, consistency, and variations between two assays of DNA fragmentation index based on acridine orange (AO) staining via AI-based fluorescence microscopy(AI-DFI), and flow cytometry (FCM-DFI) across multiple centers.
METHODS: We selected 421 male patients from Nanjing Drum Tower hospital ( Hospital G) (226 cases), Eastern Theatre General Hospital (Hospital J) (89 cases) and Jiangsu Province Hospital (Hospital S) (106 cases) . Semen samples from each patient were analyzed for routine semen parameters and for DFI using both AI fluorescence microscopy and flow cytometry. We studied the two methods\' stability as well as the correlation, consistency, and variation between the two methods\' results in various centers.
RESULTS: The two replicate studies\' results of AI-DFI and the three centers\' FCM-DFI for linear regression analysis indicated strong stability (R2>0.9).Overall(Group A), the AI-DFI results demonstrated good correlation and consistency with the FCM-DFI results of three centers (r>0.85；ICC>0.9).The semen specimens were categorized into two groups: normal specimen group (group B) and abnormal specimen group (group C) (including asthenozoospermia, oligospermia, and semen samples with high impurities).Group C\'s results showed a decline in correlation and consistency when compared to group A and group B, whereas group B\'s results showed a little rise in correlation and consistency when compared to group A. Although the consistency and correlation between the results of the two DFI testing methods in the three centers were good, there was still a significant difference between Groups A and C (P<0.05), and in Group B there was a significant difference between the two DFI testing methods only in Hospital G (p＝0.02), with no significant difference in Hospitals J and S (P> 0.05).
CONCLUSIONS: The two detection methods exhibit good stability and correlation. However, significant differences are observed in the DFI detection methods in samples with abnormal semen parameters and high complexity. The main reason for these significant differences may lie in the variations in detection principles. Each detection method has its own advantages, allowing clinical or research settings to choose between them based on laboratory conditions or specific requirements.

Tuberculosis (TB) poses a significant health threat in Taiwan, necessitating efficient detection methods. Traditional screening for acid-fast positive bacilli in acid-fast stain is time-consuming and prone to human error due to staining artifacts. To address this, we present an automated TB detection platform leveraging deep learning and image processing. Whole slide images from 2 hospitals were collected and processed on a high-performance system. The system utilizes an image processing technique to highlight red, rod-like regions and a modified EfficientNet model for binary classification of TB-positive regions. Our approach achieves a 97% accuracy in tile-based TB image classification, with minimal loss during the image processing step. By setting a 0.99 threshold, false positives are significantly reduced, resulting in a 94% detection rate when assisting pathologists, compared with 68% without artificial intelligence assistance. Notably, our system efficiently identifies artifacts and contaminants, addressing challenges in digital slide interpretation. Cross-hospital validation demonstrates the system\'s adaptability. The proposed artificial intelligence-assisted pipeline improves both detection rates and time efficiency, making it a promising tool for routine pathology work in TB detection.

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.

This study aimed to compare the color change of computer-aided design (CAD)/computer-aided manufacturing (CAM) polymethyl methacrylate (PMMA) denture teeth and conventional acrylic teeth after immersion in three staining beverages (coffee, red tea, and cola) for a day, 7 days, and 30 days.
Group 1: Conventional acrylic teeth (n = 32). Group 2: Milled CAD/CAM teeth out of PMMA disc (n = 32). The specimens of each material were further divided into four subgroups: (1) Control group, distilled water (n = 16). (2) Red tea solution (n = 16). (3) Coffee solution (n = 16). (4) Cola (n = 16). The color change ( ∆ E $\\unicode{x02206}E$ ) was assessed using a spectrophotometer at four time points: at the baseline (t0 ), on the 1st day (t1 ), on the 7th day (t2 ), and the 30th day (t3 ) of immersion. Kolmogorov-Smirnov test was applied, followed by performing independent samples t test, one-way analysis of variance and post-hoc Tukey tests to compare the color change values at different time points.
The mean score of NBS values of the coffee solution indicates perceivable color change at the end of the 30th day in the conventional acrylic teeth group. It was 0.843 ± 0.395 at t1 , then increased to 1.017 ± 0.477 at t2 and to 2.259 ± 1.059 at t3 . There is a statistically significant difference (p < 0.05) in color change values between both tooth types at the end of the 30th day of immersion in red tea solution and a statistically significant difference at the end of the 7th day (p < 0.05) and the 30th day (p < 0.05) of immersion in coffee solution.
CAD/CAM PMMA teeth are more color stable than conventional acrylic teeth after 30 days of immersion in coffee and red tea solution.