Search Results (141)

Background: Urine cytology is a highly effective, straightforward, and cost-efficient diagnostic tool for identifying neoplastic and non-neoplastic changes in the bladder, ureter, and renal pelvis. The aim of this study is to demonstrate the high sensitivity and specificity of urine cytology in detecting a wide range of urothelial lesions, including metastatic involvement. Material and Methods: Urine cytology was performed on 9639 cases between 2000 and 2025. The samples, collected from patients, were processed at the Institute of Pathology. Cytological slides were prepared using cytocentrifugation and stained with May–Grünwald–Giemsa (MGG) and Papanicolaou stains. The cytological findings were classified according to WHO, 2004 compared with histological specimens. Additionally, selected cases underwent immunohistochemical and molecular analyses. All samples were anonymized and retrospectively analyzed following the guidelines and regulations of the local ethics committee. Results: Of the total cases, 7051 were classified as benign, 1269 as malignant, and 88 as normal findings. Insufficient material was obtained in 336 cases. No complications were reported during sample collection or processing. The concordance with histological findings for neoplastic lesions was over 96%, with a false-negative rate of 1.84%. The diagnostic methods demonstrated a sensitivity of 90.7% and a specificity of 96.64%. Among the 6956 cases analyzed, 3139 were women (45.13%) and 3817 were men (54.87%). Conclusions: The diagnostic value of urine cytology in representative material is relatively high in assessing both the presence or absence of malignancy and, when applicable, the tumor grade. This large 25-year single-center review demonstrates that urine cytology retains high sensitivity and specificity for the detection of urothelial malignancy, particularly high-grade disease. However, the atypical category remains a major diagnostic challenge and contributes substantially to false-positive results. Full article

The mud crab (Scylla paramamosain), an economically important crustacean aquaculture species in southern China, is susceptible to infections due to its immune system lacking acquired immunity. An emergent disease locally termed “pus crab” has caused severe muscle lesions in pond-farmed crabs, but its etiology remained unclear. Here, we applied an integrated approach, histopathology, electron microscopy, metagenomic sequencing, and experimental infection to identify the pathogen of “pus crab”. Histological staining (H&E, Wright–Giemsa, and Masson) revealed muscle fiber dissolution, disordered fiber arrangement, and abundant interstitial spore-like bodies. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed intracellular spore morphology consistent with microsporidia. Metagenomic profiling showed a pronounced shift in the muscle microbiome, with a marked increase in microsporidian taxa at the genus level and a concurrent decline in bacterial relative abundance. Functional annotation indicated enrichment of pathways related to protein processing, ribosome biogenesis, glycosylation, and the ubiquitin–proteasome system. Isolation of spores from diseased muscle and subsequent injection into healthy crabs reproduced wild-like clinical signs and histopathology, confirming infectivity and implicating microsporidia as the likely etiological agents of “pus crab”. These findings establish a multidisciplinary framework for pathogen identification in aquaculture and provide candidate molecular and biochemical markers for early diagnosis and management. Full article

Background/Objectives: Fonsecaea pedrosoi causes chromoblastomycosis, a neglected chronic subcutaneous mycosis that remains difficult to treat. In this study, we evaluated the toxicity and the antifungal effect of [Ag(1,10-phenanthroline)₂]ClO₄ (Ag-phen) and [Ag₂(3,6,9-trioxaundecanedioate)(1,10-phenanthroline)₄]·EtOH (Ag-tdda-phen) against F. pedrosoi using in silico, in vitro and in vivo approaches. Methods: Pharmacokinetic and toxicological parameters were predicted using ADMETlab 2.0. The toxicity of the complexes was assessed using sheep red blood cells, RAW 264.7 macrophage cells, and larvae of Tenebrio molitor and Galleria mellonella. The effects of these complexes on macrophage adhesion capacity and reactive oxygen species (ROS) production were also investigated using Giemsa staining and dichlorofluorescein diacetate, respectively. In addition, their impact on the survival of G. mellonella larvae infected with conidia was evaluated. Results: Overall, computational analyses predicted favorable tolerability profiles for both complexes. In vitro assays with red blood cells and macrophages demonstrated that they exhibited selectivity indexes >10 against F. pedrosoi. These findings were corroborated by in vivo experiments in which both complexes were injected into insect larvae; the complexes demonstrating good tolerability at concentrations of up to 500 mg/L. Macrophage infection assays revealed that Ag-tdda-phen and Ag-phen markedly reduced the number of intracellular conidia. These effects appear to be associated with oxidative stress, as macrophage production of ROS significantly increased following treatment with the complexes. Furthermore, Ag-tdda-phen improved the survival of G. mellonella larvae infected with F. pedrosoi, demonstrating a protective effect. Conclusions: Collectively, our findings support the notion that silver(I)-phen derivatives represent promising candidates for the development of therapeutic options against CBM infections caused by F. pedrosoi. Full article

A herd of approximately 300 Spanish meat goats in central Alabama experienced sporadic ocular, respiratory, and reproductive diseases over two years, prompting diagnostic investigation at Auburn University’s JT Vaughan Large Animal Teaching Hospital. Five representative doelings exhibiting ocular lesions were examined. Clinical signs included conjunctivitis, corneal opacity, uveitis, and, in one severe case, systemic illness. Initial treatment with topical and systemic antibiotics provided incomplete resolution, raising suspicion of infectious keratoconjunctivitis of atypical etiology. Comprehensive diagnostic testing was performed, including aerobic and Mycoplasma cultures, Giemsa staining, and molecular assays. Moraxella bovoculi was cultured; however, Giemsa staining revealed Chlamydia elementary bodies, and a FRET-qPCR with DNA sequencing confirmed high Chlamydia pecorum loads (up to 1.1 × 10⁷ copies/swab). Mycoplasma testing was negative. Extended treatment with systemic and topical oxytetracycline led to gradual clinical improvement, with C. pecorum DNA declining over 22,000-fold and becoming undetectable after five weeks. This case represents the first documented report of C. pecorum–associated keratoconjunctivitis in goats in the United States. The findings underscore the diagnostic importance of molecular assays for detecting intracellular pathogens that may be missed by culture. The protracted treatment course highlights the therapeutic challenges posed by chlamydial infections due to their intracellular persistence. Additionally, the concurrent detection of M. bovoculi suggests the potential for mixed infections influencing disease severity. These results emphasize C. pecorum as an emerging pathogen of caprine ocular disease with implications for herd health and management. Full article

This research aimed to establish reference intervals and evaluate the influence of age in 56 Nguni cattle raised in Mozambique. Blood samples containing EDTA as anticoagulant were collected. The erythrogram and total leukocyte count were analyzed using the BC-2800 Vet Mindray^® automatic counter. The differential counting of leukocytes was performed in blood smears stained using Giemsa and Mcgruwald’s stain. Statistical analysis was performed using the statistical analysis system (SAS). Analysis of variance was performed using the GLM procedure, and mean contrasts were analyzed using Duncan’s parametric test at 5% significance, with the Shapiro–Wilk and Levene tests for date normalities. The reference intervals for the erythrogram are as follows: red blood cells: 6.78 to 7.40 × 10¹²/L; hemoglobin: 10.77 to 11.36 g/dL; hematocrit: 28.02 to 29.56%; mean corpuscular volume (MCV): 39.91 to 43.02 fL; mean corpuscular hemoglobin (MCH): 15.27 to 16.44 pg; mean corpuscular hemoglobin concentration (MCHC): between 37.86 and 39.10 g/dL; and red blood cell distribution width (RDW): between 16.98 and 19.40%. For leukograms, the following references values were obtained: total leukocytes: between 14,106 and 16,233 × 10⁶/L; basophils: between 32 and 165 × 10⁶/L; eosinophils: between 823 and 1262 × 10⁶/L; band neutrophils: between 25 and 87 × 10⁶/L; segmented neutrophils: between 2510 and 3249 × 10⁶/L; total neutrophils: between 2565 and 3306 × 10⁶/L; lymphocytes: between 9471 and 11,474 × 10⁶/L; and monocytes: between 154 and 296 × 10⁶/L. Age influenced MCV, MCH, leukocytes, eosinophils, and lymphocytes. Leukogram reference intervals of other countries could not be used for the breed of Mozambique without making gross errors. Full article

Helicobacter pylori is a gastric pathogen that induces chronic gastritis, which may progress to neutrophilic activity, glandular atrophy, intestinal metaplasia, and gastric carcinoma. The aim of this study was to evaluate H. pylori-induced tissue damage. A total of 602 gastric biopsy samples were collected, categorized, and analyzed using hematoxylin and eosin and Giemsa staining, followed by molecular confirmation through PCR targeting the species-specific 16S rRNA gene. H. pylori density and histopathological features were evaluated and graded according to the updated Sydney classification system. H. pylori was detected in 55% (n = 334) of cases, and the antrum (50.83%, p < 0.00001) was the predominant site. A slightly higher prevalence was observed in females, accounting for 56.9% compared to males at 43.1%, which was attributed to sociocultural exposure differences. Individuals aged 11–40 years accounted for 58.3% (n = 195), highlighting early-age acquisition of infection. H. pylori infection was significantly linked to moderate-to-severe inflammation (63.2%, p < 0.00001) and neutrophilic activity (53.3%, p < 0.00001). Intestinal metaplasia and atrophy were infrequent, present in 0.6% (95% CI, 0.02, p = 0.149) and 0.9% (95% CI, 0.05, p = 0.430) of individuals. H. pylori infection causes chronic inflammation and neutrophilic infiltration of the stomach mucosa. Early identification and histopathological examination are essential in assessing H. pylori-related gastric pathology. Full article

Trypanosomiasis significantly impacts camel health and productivity, posing a major challenge to food security in regions with large camel populations. In this study, we investigated the microscopic and molecular prevalence, performed phylogenetic analysis, and explored risk factors associated with Trypanosoma evansi (T. evansi) infection in 400 randomly selected suspected camels (Camelus dromedarius) from 10 districts of Punjab, Pakistan. Blood samples were collected for microscopic examination of Giemsa/Field’s-stained smears, and three PCR primer sets (ITS1CF/BR, pMUTec, RoTat 1.2) were used to detect the presence of T. evansi. PCR-based prevalence was higher (14.8%; CI 11.4–18.6) as compared to the microscopic examination (8.3%; CI 5.7–11.4) of samples. The targeted primers amplified DNA fragments of 210, 205, and 478 base pairs, respectively. Phylogenetic analysis showed 100% homology between local isolates and those from India, Sudan, Malaysia, Egypt, and Kenya. Risk analysis identified female gender (OR 2.1) and being in Southern Punjab (OR: 1.9) as significant factors associated with disease. Significantly (p < 0.05) reduced total protein (5.51 ± 0.05), albumin (2.77 ± 0.04), and globulin (2.57 ± 0.06) levels were found in PCR-positive camels. This study provides new molecular and phylogenetic data on T. evansi in Pakistan. Full article

Malignant pleural effusion, commonly referred to as MPE, is a prevalent complication associated with individuals diagnosed with neoplastic disorders. The data acquired by pleural fluid cytology is beneficial for diagnostic objectives. Consequently, the initial step in the diagnostic procedure for lung cancer is the analysis of pleural effusion fluid. This research aims to provide a cutting-edge model for analyzing PE cytology images. This model utilizes a computer-aided diagnosis (CAD) system that integrates hyperspectral imaging (HSI) technology for the classification of spectral variations. Giemsa, which is one of the most popular microscopic stains, is employed to stain the samples, after which a sensitive CCD mounted on a microscope captures the images. Subsequently, the HSI model is tasked with obtaining the image spectra. Principal Component Analysis (PCA) constitutes the concluding phase in the classification procedure of various cell types. We expect that the suggested technique will enable medical professionals to stage lung cancer more rapidly. In the future, we aspire to develop an extensive data system that utilizes deep learning techniques to facilitate the automatic classification of cells, thereby ensuring the most precise diagnosis. Furthermore, enhancing accuracy and minimizing data dimensions are important priorities to accelerate diagnostics, conserve resources, and reduce computing time. Full article

Background: Patients with chronic lymphocytic leukemia (CLL) who were not receiving treatment were included in this experimental prospective correlation study. We aimed to elucidate the complex relationship between smudge cells, surface CD20, and soluble CD20 in CLL patients. Methods: We created blood smears from blood samples collected from our patients using a manual technique consistently performed by the same technician. The May–Grunwald Giemsa dye was used to stain all of the slides. The B-cell phenotypic was analyzed using the FacsCanto II flow cytometer (Becton Dickinson, CA, USA) at the time of diagnosis. Competitive Enzyme-Linked Immunoassay (ELISA) was used to quantitatively assess the amounts of soluble CD20/MS4A1. Results: The percentage of smudge cells and soluble CD20 antigen levels were shown to be significantly inversely correlated, suggesting a considerable link (correlation coefficient (r) = −0.51, p = 0.006). Similarly, a significant inverse relationship (r = −0.36, p = 0.04) was found by the Spearman correlation test between the smudge cell ratio and CD20 median fluorescence intensity (MFI) on cell surfaces. Soluble CD20/MS4A1 and surface CD20 MFI were shown to have a weakly positive association that was almost statistically significant (Spearman’s rho = 0.34, p = 0.064). With a sensitivity of 69% and specificity of 86%, we discovered that a cut-off value of 2.2 ng/dL for soluble CD20 predicted higher smudge cells (area under the curve (95% confidence interval (CI)): 0.75 (0.57 to 0.93), p = 0.021). Conclusions: We found a significant inverse association between smudge cells and both surface CD20 and soluble CD20/MS4A1 in our study examining the correlation between smudge cells, soluble CD20, and CD20/MS4A1 in CLL patients. Our findings indicate that soluble CD20 may contribute to understanding the pathophysiology of smudge cells and could be further investigated as a potential prognostic marker in CLL. Full article

Background: Erythroblasts have recently been identified as host cells for malarial parasites, revealing a previously underappreciated host–parasite interaction. However, their extremely low abundance in peripheral blood has hindered progress, especially in elucidating the biological significance of parasitized erythroblasts (pEBs) in vivo. Methods: Here, we visualized pEBs in a murine model and established a method to increase their number in peripheral blood by immunizing mice with live Plasmodium yoelii 17XNL, followed by challenge with P. berghei ANKA. Results: Immunized mice were protected from cerebral malaria and survived longer, during which pEBs appeared in circulation and were detected using Giemsa-stained smears. All blood-stage parasite forms were identified within pEBs, including enucleating erythroblasts. Conclusions: This model enables in vivo/ex vivo analysis of pEB biology without bone marrow/spleen isolation, thus lowering technical/ethical barriers for the field. Full article

Background: Phosphaturic mesenchymal tumours secreting fibroblast growth factor 23 (hereinafter referred to as FGF23+ PMT) are rare neoplasms that can cause hypophosphataemic osteomalacia, owing to excessive FGF23 production. Mast cells (MCs) play a key role in tumour biology by modulating proliferative activity of atypical cells, resistance to innate and acquired immunity, angiogenesis, and metastatic behaviour. However, MCs associated with FGF23+ PMT have not previously been investigated. This study, to our knowledge, is the first to characterise features of the tumour microenvironment through spatial phenotyping of the immune and stromal landscape, together with histotopographic mapping of intercellular MC interactions with other subcellular populations in FGF23+ PMT. Methods: Histochemical staining (haematoxylin and eosin, toluidine blue, Giemsa solution, picro-Mallory protocol, silver impregnation), as well as monoplex and multiplex immunohistochemical staining with spatial phenotyping, were performed to detect atypical FGF23-secreting cells, immune cells (CD3, CD4, CD8, CD14, CD20, CD38, CD68, or CD163), stromal components (CD31, α-SMA, or vimentin), and specific MC proteases (tryptase, chymase, or carboxypeptidase A3). Bioinformatics analysis using artificial intelligence technologies was applied for spatial profiling of MC interactions with tumour, immunocompetent, and stromal cells in the tumour microenvironment. Results: Bioinformatic analysis of the entire tumour histological section, comprising over 70,000 cells stained using monoplex and multiplex immunohistochemical protocols, enabled identification of more than half of the cell population. The most abundant were CD14+ (30.7%), CD163+ (23.2%), and CD31+ (17.9%) cells. Tumour-associated MCs accounted for 0.7% of the total pool of immunopositive cells and included both mucosal and connective tissue subpopulations, predominantly of the tryptase + chymase-CPA3-specific protease phenotype. This pattern reflected combined multidirectional morphogenetic processes in the patient’s FGF23+ PMT. More than 50% of MCs were colocalized with neighbouring cells of the tumour microenvironment within 20 μm, most frequently with monocytes (CD14+CD68+), M2 macrophages (CD68+CD163+), and endothelial cells (CD31+). In contrast, colocalization with atypical FGF23-secreting cells was rare, indicating minimal direct effects on tumour cell activity. Interaction with T lymphocytes, including CD8+, was also infrequent, excluding their activation and the development of antitumour effects. Mapping of MC histotopography validated the hypothesis of their inductive role in monocyte differentiation into M2 macrophages and probable polarisation of macrophages from M1 into M2, thereby contributing to slow tumour growth. MCs were further involved in extracellular matrix remodelling and participated in the formation of pro-osteogenic niches within the FGF23+ PMT microenvironment, leading to pathological osteoid development. Conclusions: This study demonstrated active MC participation in the evolution of the FGF23+ PMT microenvironment. The findings may be applied in translational medicine to develop novel algorithms for personalised therapy in patients with FGF23-secreting tumours, offering an alternative when surgical removal of the tumour is not feasible. Full article

The family Anaplasmataceae comprises etiological agents of infectious diseases of significant importance. This study aimed to achieve the in vitro isolation and propagation of an Anaplasma sp. using tick-derived cell lines. The study was realized in Seropédica municipality, Rio de Janeiro, Brazil. Blood smears from a naturally infected bovine revealed cytoplasmic inclusions in blood cells. To isolate and propagate the organism, IDE8 and ISE6 tick cell lines derived from Ixodes scapularis were used. Two methods of inoculum preparation were employed: Histopaque^® density gradient and platelet-rich plasma separation. Following infection, cells were maintained in L-15B medium without antibiotics at 34 °C, and infection was monitored weekly by Giemsa-stained cytocentrifuge smears. After achieving ≥ 70% infection, bacteria were subcultured and successfully cryopreserved and resuscitated. PCR amplification and sequencing of 16S rDNA, 23S rDNA, rpoB, and groEL genes were performed for molecular characterization. Phylogenetic analyses revealed that the isolated strain clustered within the A. platys-like clade. This study reports the successful in vitro isolation, propagation, and cryopreservation of the ‘A. platys-like strain Natal’ bacterium in tick cell lines and provides molecular evidence supporting its phylogenetic classification. These findings contribute to the understanding of genetic variability and host–cell interactions of Anaplasma spp., laying the groundwork for future research. Full article

Background/Objectives: Platelet counts can be affected by storage conditions, potentially leading to pseudothrombocytopenia. The present study aimed to investigate temperature-dependent changes in platelet counts and morphology in whole blood samples anticoagulated with heparin or EDTA. We also examined the molecular mechanism of cold-induced aggregation via integrin GPIIb/IIIa–fibrinogen interaction using established bioinformatics technologies (docking simulation). Methods: Peripheral blood was collected from healthy volunteers (n = 6) and treated with either heparin or EDTA. The samples were stored at 4 °C, room temperature, or incubated at 37 °C. Platelet counts were measured using an automated hematology analyzer. The morphology of various blood cells in smears was assessed using the May-Grünwald Giemsa staining method. Docking simulations using an available software (HADDOCK 2.4) were performed to evaluate integrin–fibrinogen binding at different temperatures. Results: In automated blood cell counting, platelet counts in heparinized blood were significantly decreased under low-temperature conditions (4 °C), but this decrease was restored to levels comparable to those at room temperature upon warming to 37 °C (p < 0.05). No significant changes were observed in EDTA-treated samples. Microscopical findings showed platelet aggregation only in heparinized samples at 4 °C, with normal morphology restored upon warming (37 °C). Docking simulations estimated stronger integrin GPIIb/IIIa–fibrinogen binding at 4 °C than at 37 °C (p = 0.0286), suggesting temperature-dependent enhancement of molecular interactions. Conclusions: These findings indicate that heparin can induce reversible platelet aggregation at low temperatures in whole blood samples, leading to pseudothrombocytopenia. This phenomenon may be mediated by increased integrin GPIIb/IIIa–fibrinogen binding. Full article

Background: The microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections; however, this technique is time consuming and requires experienced and well-trained operators. Therefore, there is a growing interest in molecular diagnostic techniques, including commercial PCR assays. The aim of this multicentric study was to evaluate a commercial real-time PCR for the detection of intestinal protozoa in fecal samples. Methods: The samples were routinely examined using conventional techniques, such as macro- and microscopic examination after concentration, Giemsa or Trichromic stain, Giardia duodenalis, Entamoeba histolytica/dispar or Cryptosporidium spp. antigens research, and amoebae culture. The samples were frozen by the participating laboratories, retrospectively extracted and examined with one-step real-time PCR multiplex using the Allplex™ GI-Parasite Assay (Seegene Inc., Seoul, Korea). Results: A total of 368 samples were analyzed from 12 Italian laboratories. Compared to traditional techniques, the sensibility and specificity of the real-time PCR kit were as follows: 100% and 100% for Entamoeba histolytica, 100% and 99.2% for Giardia duodenalis, 97.2% and 100% for Dientamoeba fragilis, and 100% and 99.7% for Cryptosporidium spp., respectively. Conclusions: The Allplex™ GI-Parasite Assay exhibited excellent performance in the detection of the most common enteric protozoa. Full article

Small rodents are known hosts of various pathogens, including Hepatozoon, but until now, in Brazil, only Hepatozoon milleri has been described in these animals. In this study, liver samples and blood smears were obtained from 289 rodents belonging to 14 Cricetidae and two Muridae species that had been captured in municipalities of the states of Paraná and Rio de Janeiro. Smears were stained with Giemsa, and gametocytes were detected via microscopy in 10.72% (n = 31/289) of samples, with these individuals representing three rodent species. Significant morphometric differences were observed in gametocyte measurements in Akodon rodents. Using conventional PCR, Hepatozoon spp. 18S rDNA fragments were amplified in 24.91% (n = 72/289) of samples, with those individuals representing seven rodent species. Phylogenetic analyses clustered 41 sequences from this study into a subclade with other sequences from small mammals in Brazil, identifying four distinct haplotypes, and, for the first time, a relationship between Hepatozoon haplotype and gametocyte length was observed. Based on phylogenetic analysis, this study reinforces the trophic relationship between rodents and reptiles as a possible link in the Hepatozoon transmission cycle in South America. Furthermore, our findings expand knowledge on Hepatozoon spp. hosts, describing Oxymycterus nasutus and Oxymycterus quaestor as new host species and identifying two novel circulating haplotypes in rodents from Paraná State, southern Brazil. Full article