Search Results (2,678)

Background/Objectives: The traditional classification of astrocytes was based on morphological differences between astrocytes and their location in brain tissue. Astrocytes stained by impregnation techniques were divided into protoplasmic and fibrous astrocytes. We still use this classification, often supplemented by GFAP immunostaining. However, protoplasmic astrocytes have been found in the human cerebral cortex as GFAP-negative cells. Methods: In this study, astrocytes were identified using Cajal’s gold sublimation method and GFAP immunostaining. Biopsy samples of normal brain tissue (n = 25) were obtained from adult patients diagnosed with traumatic brain injury, stroke, gliomas and brain metastases. Results: In all samples, GFAP-positive fibrous astrocytes were found in the subpial region (layer I-I) and in the white matter. GFAP-positive protoplasmic astrocytes were absent or occurred only rarely in the cortical gray matter (layer III–VI) in samples from patients diagnosed with a tumor. Similar staining was also observed using the Cajal method. However, in samples from patients with traumatic brain injury accompanied by high intracranial pressure, GFAP-positive areas with numerous astrocytic processes and cells with a morphology similar to protoplasmic astrocytes were found. Conclusions: We can conclude that protoplasmic astrocytes are GFAP-negative cells that respond to brain injury by GFAP expression. We consider this finding to be a sign of protoplasmic astrocyte differentiation. On the other hand, fibrous astrocytes are GFAP-positive and respond to brain injury with increased GFAP expression. These results raise questions regarding the classification of astrocytes and, in particular, the histological visualization of the neuro-glial-vascular unit. Full article

Postharvest diseases caused by fungal pathogens lead to significant economic losses in citrus production. The intensive use of synthetic fungicides has triggered the emergence of resistant strains and environmental contamination, emphasizing the need to search for sustainable alternatives. This study evaluated the antifungal efficacy of a strawberry leaf aqueous extract (SLE) against local isolates of citrus phytopathogens such as Penicillium digitatum and P. italicum (both fungicide−sensitive and fungicide−resistant) and Geotrichum citri-aurantii. In vitro assays showed complete inhibition of mycelial growth for all pathogens on potato dextrose agar plates supplemented with 0.05 g·mL⁻¹ SLE after 5 days at 24 °C. Furthermore, total inhibition of conidial germination and loss of viability were achieved at 0.1 g·mL⁻¹ following an 8 or 24 h exposure period respectively. To elucidate the underlying mode of action, membrane integrity and cellular ultrastructure were examined. SYTOX™ Green staining revealed increased membrane permeability, and transmission electron microscopy showed marked intracellular disorganization in SLE-treated conidia from all phytopathogens. These findings were further validated through in vivo assays using artificially inoculated lemons where a significant reduction in green mold incidence was demonstrated. Overall, SLE exhibited broad-spectrum efficacy against major citrus postharvest pathogens, effectively overcoming established fungicide resistance. Our results could position SLE as a promising biocontrol agent for sustainable fruit preservation. Full article

Background/Objectives: Conventional chordomas are rare malignant bone tumors characterized by brachyury expression. The presence of polygonal-to-epithelioid neoplastic cells containing rich cytoplasm and relatively large nuclei in a myxoid background is a characteristic cytological feature of conventional chordomas. Physaliphorous cells, characterized by the presence of multiple well-marginated vacuoles within the cytoplasm, are also characteristic of this rare bone tumor. However, the cytological diagnosis of conventional chordomas can be challenging because of their rarity and lack of specific features. Therefore, useful immunocytochemical markers are required. Immunohistochemical staining for brachyury has been widely used for diagnosis; however, immunocytochemical staining using cytological specimens has not yet been performed. The purpose of this study was to evaluate the usefulness of the immunocytochemical staining for brachyury in cytodiagnosis of conventional chordoma. Methods: This study included patients diagnosed with conventional chordoma based on postoperative histopathological findings who underwent intraoperative squash cytological examination. Cytological features and immunocytochemical staining for brachyury were evaluated. Results: Two patients with conventional chordoma were included. The tumor extended from the lumbar spine to the cauda equina and clivus. Cytological examination revealed the presence of small sheets and isolated polygonal neoplastic cells with rich cytoplasm and round nuclei with inconspicuous nucleoli in a myxoid background. Cellular clusters were noted in one specimen and physaliphorous cells were observed in the other. Immunocytochemical staining for brachyury revealed positive nuclear expression in both specimens. Conclusions: Immunocytochemical staining for brachyury could provide useful information for the cytodiagnosis of this rare tumor, even in cases lacking typical cytological features. Full article

To study the soil moisture dynamics and rainfall infiltration characteristics of karst sloping farmland and their driving factors, an abandoned farmland was selected for this study, and five monitoring points (from the foot, S1, of the slope to the top, S5) were set along the terrain gradient. The volumetric water content data of the 0–40 cm soil layer was obtained through in situ monitoring for one year. The infiltration characteristics were quantified in combination with a staining tracer test, and the soil properties were determined. The results showed that the soil moisture content increased with the deepening of the soil layer, and there was significant slope differentiation. The moisture content in the downhill slopes (S1, S2) was significantly higher than that in the uphill slopes (S4, S5), and the annual average value of S5 was 27.4% lower than that of S1. The moisture difference (Δθ, the difference in moisture content between hillslope and flatland) changed from positive to negative from the foot of the slope to the top, indicating that moisture was transported downward along the slope surface. A dye tracer showed that from S1 to S5, the water transport pathway gradually shifted from exhibiting deeper vertical penetration and narrower lateral spread to showing shallower vertical penetration and wider lateral spread. The preferential flow index decreased from 46.6 ± 2.3% to 34.7 ± 2.1%, indicating a progressive reduction in rapid vertical channeling, while the lateral flow index reached its peak (21.4 ± 2.7%) in the middle of the slope (S3), suggesting enhanced horizontal water redistribution at this position. Correlation analysis indicated that soil bulk density was extremely significantly negatively associated with infiltration capacity, while capillary porosity, non-capillary porosity, total porosity, organic matter, and high aggregate content were extremely significantly positively associated with infiltration capacity. These results revealed that the topographic gradient affected soil moisture and water infiltration paths by regulating soil physical properties in this karst forest ecosystem. It should be noted that the research results are only applicable to one slope and should not be directly extended to all karst slope agricultural landscapes. Full article

Background/Objectives: Tumor-infiltrating lymphocytes (TILs) are recognized predictors of response to neoadjuvant chemotherapy (NACT) and prognosis in breast cancer, particularly in triple-negative (TN) and HER2-positive subtypes. However, the additional predictive value of morphological features of the inflammatory infiltrate beyond TIL quantification is not fully established. We aimed to assess the predictive value of TILs for response to NACT in breast cancer and to determine whether morphological characteristics of the inflammatory infiltrate enhance predictive accuracy. Methods: We analyzed 477 patients with stage II–III breast cancer treated with NACT between 2009 and 2016. Diagnostic core needle biopsies were prospectively re-evaluated. TILs were quantified according to International TILs Working Group recommendations. Morphological features of the infiltrate, including cell composition (lymphocytic vs. plasma cell-rich), heterogeneity, and localization, were evaluated using standardized criteria. Pathologic complete response (pCR) was defined as absence of invasive tumor in the breast and in the axillary lymph nodes (ypT0/Tis ypN0). Univariate and multivariate logistic regression analyses were performed to assess the predictive value of TILs (quantitative and morphological assessment) to achieve pCR for the entire cohort and by surrogate molecular subtype. Results: A TIL cutoff of >20% was identified as optimal for predicting pCR. High TILs were significantly associated with high-grade tumors, elevatedKi67, HER2-positive and TN subtypes, presence of plasma cells, and intraepithelial and heterogeneous infiltrates. In the overall cohort, TILs > 20% significantly increased the likelihood of pCR (OR 3.9, 95%IC 2.5–6.0, p < 0.001) and was an independent predictor of pCR. A combined variable incorporating TIL level and homogeneity improved predictive performance, with homogeneously high TILs emerging as a strong predictor of pCR (OR 5.521, 95%CI 3.174–9.603, p < 0.01). Plasma cell-rich and intraepithelial infiltrates were also associated with higher pCR rates (respectively, OR 2.7, 95%CI 1.5–5.0, p = 0.001 and OR 2.8, 95%CI 1.6–5.0, p < 0.001). Subtype-specific analyses confirmed the predictive value of TILs in TN tumors, but not in HER2-positive ones. Notably, in luminal B-like tumors, high TILs were the only independent predictor of response (OR 17.982, 95%CI 3.115–103.815, p = 0.001). Conclusions: TIL assessment on routine H&E-stained biopsies is a robust predictor of response to NACT in breast cancer that is readily available, cost-neutral and does not require additional techniques. Integration of simple morphological features significantly enhances predictive accuracy and may refine treatment stratification, particularly in luminal B-like tumors. Full article

Background/Objectives: Glioblastoma multiforme (GBM) treatment is limited by tumor hypoxia and poor specificity of therapeutic agents. To address these challenges, we developed brain-targeted liposomes co-encapsulating 5-aminolevulinic acid (5-ALA) and catalase (CAT), termed brain-targeted 5-ALA-CAT liposomes (BACL), which were surface-modified with the Angiopep-2 ligand to enhance blood–brain barrier penetration and achieve multimodal therapy combining targeted delivery and oxygen generation. Methods: BACL was prepared and characterized. Tumor targeting was verified by flow cytometry and in vivo imaging. In vitro antitumor activity was evaluated by wound-healing assay, colony formation assay, live/dead staining, MTT assay, and Western blotting. In vivo efficacy, apoptosis, and safety were assessed in a subcutaneous xenograft model. Transcriptome sequencing and qRT-PCR were employed to identify molecular mechanisms and novel targets. Results: BACL exhibited favorable physicochemical properties (size: 122.4 nm, PDI: 0.189, zeta potential: −12.3 mV) and spherical morphology as observed by TEM, with encapsulation efficiencies of 51.2% for 5-ALA and 43.8% for CAT. Compared with unmodified 5-ALA, BACL increased the cellular uptake efficiency by 1.6-fold in glioma cells while maintaining catalytic stability for sustained oxygen generation. In vitro experiments demonstrated that BACL significantly inhibited glioma cell migration, colony formation, and cell viability, and induced apoptosis. In a subcutaneous xenograft tumor model, BACL-mediated photodynamic therapy (PDT) achieved a tumor growth inhibition rate of 52%, with apoptosis induction via regulation of Bcl-2, Bax, and p53 expression, and no obvious toxicity to major organs was observed. Transcriptomic analysis combined with qRT-PCR validation revealed that BACL activates multiple antitumor signaling pathways, including targeted inhibition of IL-10 and CXCL13 to disrupt cytokine–receptor interactions, as well as coordinated regulation of S100A3 and IGSF-9 expression to suppress glioma progression. Conclusions: These multimodal actions enhanced PDT efficacy while remodeling the tumor microenvironment. Our findings position BACL as a promising therapeutic platform integrating targeted delivery, hypoxia alleviation, and immunomodulation for GBM therapy. Full article

Background and Clinical Significance: Cardiac and mediastinal angiosarcomas are rare, aggressive malignancies that often present with nonspecific symptoms and pose significant diagnostic challenges. Tumor heterogeneity and necrosis may lead to false-negative biopsy results; Case Presentation: We report a 64-year-old man who initially presented with cardiac tamponade of unclear etiology. Despite an extensive workup, the patient remained asymptomatic for five months before re-presenting with dyspnea and a large mediastinal mass compressing the right heart, along with a lytic rib lesion. Initial ultrasound-guided biopsy of the rib lesion demonstrated a benign vascular proliferation consistent with Masson tumor (intravascular papillary endothelial hyperplasia), which was discordant with aggressive imaging findings. Further evaluation with positron emission tomography–computed tomography (PET-CT) revealed peripheral metabolic activity, and cardiac magnetic resonance imaging (MRI) demonstrated a heterogeneous mass with central necrosis and peripheral enhancement. A repeat CT-guided biopsy targeting the metabolically active region confirmed angiosarcoma, with immunohistochemical staining demonstrating diffuse positivity for ERG, CD31, and CD34. The patient was treated with palliative radiation and paclitaxel-based chemotherapy but experienced rapid clinical decline and transitioned to comfort-focused care; Conclusions: This case highlights the importance of correlating imaging with pathology and emphasizes the risk of sampling error in necrotic tumors. PET-guided biopsy targeting viable tumor regions is essential in cases with discordant findings. Full article

The bidirectional differentiation potential of skeletal muscle satellite cells (SMSCs) enables them to differentiate into myofibers or intramuscular adipocytes, which affects meat quality in livestock. However, how insulin regulates ovine SMSC metabolism remains poorly understood. SMSCs were isolated from the longissimus dorsi muscle of 1-day-old Hu sheep, cultured, identified, and induced to differentiate with insulin. After induction, lipid droplet formation and the number of nuclei per cell were assessed, and samples were collected before adipogenic induction (No_AD) and after adipogenic induction (AD) for qPCR and whole-transcriptome sequencing. Immunofluorescence confirmed cells were positive for PAX7 and DESMIN. Bodipy, Oil Red O, and hematoxylin staining revealed lipid droplets and multinucleated cells. Sequencing and qPCR indicated that insulin promoted fatty acid uptake and utilization, inhibited adipogenic differentiation, and promoted myogenic differentiation. Integrated ceRNA analysis suggested that miR-2447-z and MSTRG.8123.1 may coordinate muscle development and lipid metabolism. In conclusion, under insulin induction, ovine SMSCs may undergo metabolic adaptation through the ceRNA network mediated by miR-2447-z and MSTRG.8123.1, exhibiting enhanced myogenesis, suppressed adipogenesis, and lipid droplet accumulation. These findings provide new insights into insulin-regulated SMSC metabolism, suggesting that leveraging the bidirectional differentiation potential of SMSCs to in-fluence muscle characteristics and fat deposition may be a feasible approach for im-proving meat production traits in sheep. Full article

Introduction: The genus Thorichthys comprises nine species of Central American freshwater cichlids, which are distributed along the eastern slopes of Mexico and Honduras. These species are grouped into two subgroups: “helleri” and “meeki”. While some analyses of the group’s ecomorphology and character evolution have been conducted, the skeletal structures of this genus remain to be described. Objective: This study provides an osteological description of the species in the genus Thorichthys. Methodology: The specimens were sectioned and stained with alizarin red, and the bone structures were analyzed and described. Results: The genus Thorichthys possesses 12 abdominal vertebrae and 12–13 caudal vertebrae. Species of “helleri” group are characterized by the following points: (1) there is a urohyal bone with a sharply concave dorsal margin and a spiniform, inclined process; (2) typically, there are a total of 14–15 gill rakers in the first gill arch; (3) the opercular bone bears a dorsally displaced, posterior protuberance; (4) a total of 12–13 anal pterygiophores; and (5) a slightly recurved cephalic profile. Meanwhile species of the “meeki” group exhibit the following characteristics: (1) a urohyal with a broad, unsharpened dorsal concavity and a small, blunt, nearly straight process; (2) a total of 15–21 gill spines; (3) a dorsally positioned protuberance in the middle of the opercular bone; (4) a total of 14–16 proximal anal pterygiophores; and (5) a straight cephalic profile. Conclusions: These bony elements are proposed as a complement to the taxonomic differentiation of the species in Thorichthys, and to contribute new morphological characteristics to the study of other cichlid genera. Full article

Background: Metastases to the uterine cervix from extragenital malignancies represent uncommon clinical events, with breast invasive lobular carcinoma (ILC) documented as the predominant primary source in reported literature. Objectives/Aim: To characterize the clinicopathologic features of ILCs metastatic to the uterine cervix. Methods: We performed a PubMed search using several keywords. Results: A total of 29 studies were included in the final analysis. The mean age at presentation of cervical metastasis was 56.8 ± 2.0 years. The mean interval between the initial diagnosis of ILC and the detection of cervical metastasis was 55.6 ± 8.2 months. Clinical presentations included vaginal bleeding, pelvic pain, and unhealthy enlarged, indurated uterine cervix on local examination. The diagnosis was established via tissue biopsy and immunohistochemical stains (positive reactivity for CK7, ER, PR, E-Cadherin, GATA3, GCDP-15 and mammaglobin). There are no consensus treatment protocols, and therapy should be tailored individually based on the extent of disease. Combined surgical and systemic therapy was the most commonly used modality. Conclusions: Metastasis of breast ILCs to the uterine cervix poses a significant diagnostic challenge. A high index of clinical suspicion and detailed clinical history are essential for accurate diagnosis. Full article

Background/Objectives: DNA-targeting small molecules that induce replication stress represent a promising strategy in anticancer drug development. 1,8-Naphthalimide (NI) derivatives are well-established DNA-intercalating agents, and heterocyclic annulation offers a rational approach to enhancing their potency and tumor selectivity. Here, we report the synthesis and biological evaluation of a novel series of benzofuran-containing naphthalimide derivatives, with particular focus on the lead dinitro-substituted compound 5d. Methods: Cytotoxic activity was assessed using the MTT assay in A549 (p53 wild-type), H1299 (p53-null), and MRC-5 cells. Long-term antiproliferative effects were evaluated by clonogenic survival assay. Cell cycle distribution was analyzed by propidium iodide staining and flow cytometry. Replication stress and DNA damage were quantified by EdU incorporation and γH2AX immunofluorescence, respectively. Apoptosis was assessed by Annexin V/PI staining and caspase-3/7 activation assay. p53 nuclear accumulation and autophagy induction were evaluated by immunofluorescence and Western blot, using LC3 as an autophagic marker. Results: All compounds exhibited cytotoxic activity in the nanomolar range, with 5d emerging as the most potent and selective. Clonogenic survival was significantly reduced, indicating durable suppression of proliferative capacity. Treatment with 5d induced G₁ arrest in A549 cells and the accumulation of H1299 cells in G₂/M, consistent with p53-dependent and p53-independent checkpoint activation, respectively. EdU incorporation was markedly reduced, while γH2AX intensity increased, collectively supporting a replication stress-driven mechanism of DNA damage. Apoptosis was confirmed by increased Annexin V-positive populations and caspase-3/7 activation. LC3 puncta formation and LC3-I/LC3-II conversion were increased, indicating LC3 processing and autophagosome accumulation consistent with the activation of autophagy-related processes. Conclusions: 5d induces a cellular phenotype consistent with replication stress, including reduced EdU incorporation, γH2AX accumulation, cell cycle arrest, and apoptotic cell death in a p53 status-dependent manner. These findings establish benzofuran-annulated naphthalimides as a promising scaffold for the development of anticancer agents that exploit replication stress vulnerabilities in tumor cells. Full article

Introduction: The liver is a central metabolic regulator responsible not only for energy storage and allocation but also for the production of essential egg proteins. In seasonal breeders such as brown trout, physiological demands increase during spawning, requiring strong metabolic and structural adaptations. Understanding the cellular mechanisms underlying these fluctuations is essential for clarifying how liver function is regulated during reproduction. Objective: The aim of this study was to investigate cellular mechanisms underlying liver remodelling during the reproductive cycle of male and female brown trout by examining proliferation and apoptosis markers at both molecular and protein levels during four distinct reproductive stages: spawning capable, regressing, regenerating, and developing. Methodology: Adult male and female brown trout were sampled at four reproductive stages. Liver gene expression of proliferating cell nuclear antigen (PCNA) and caspase-3 was analysed using qRT-PCR. At the protein level, liver tissue sections were immunolabelled with antibodies against PCNA and caspase-3. Caspase-3 staining intensity was quantified using ImageJ, while PCNA expression was assessed by counting positively stained nuclei. Results: PCNA gene expression was upregulated during the regressing stage, while PCNA-positive nuclei immunolabelling increased during the regressing and spawning-capable stages. Caspase-3 transcript levels showed no significant differences among stages, whereas immunostaining increased during regenerating and developing stages. Conclusions: Overall, the liver undergoes dynamic cellular remodelling throughout the reproductive cycle, balancing proliferation and apoptosis to meet reproductive demands. The results suggest that proliferation is more evident during regenerating stages, while apoptotic activity may be regulated post-transcriptionally. PCNA also indicates that proliferative activity may persist during spawning capable stages, reflecting ongoing mitotic readiness despite reduced overall proliferation. Full article

Fiber-degrading microorganisms are widely recognized for their potential to convert renewable lignocellulosic biomass into animal feed. However, translating this potential into practical application faces five critical yet underappreciated challenges. First, current screening methods, primarily including plate dilution and Congo red staining, are low-throughput, poorly reproducible and fail to capture the synergistic actions of natural microbial consortia. Second, the lack of standardized assays for quantifying cellulolytic activity compromises the reliability of cross-study comparisons. Third, safety assessments for fiber-degrading microorganisms remain superficial, with most studies neglecting mycotoxin production, antibiotic resistance gene transfer and long-term colonization risks. Fourth, fundamental differences between fungal and bacterial degradative systems, such as enzyme multiplicity, oxygen requirements and cellulosome assembly, are rarely considered in strain selection, leading to suboptimal application outcomes. Finally, the vast majority of positive in vitro degradation results fail to translate into improved animal performance in vivo, owing to poor microbial survival in the gastrointestinal tract, mismatched enzyme activity with gut pH and temperature, coupled with the absence of dose–response validation. This review critically evaluates these five bottlenecks across fiber-degrading microorganism types, screening platforms and practical livestock production applications. Overall, future progress should depend less on discovering “novel” strains and more on establishing standardized screening pipelines, rigorous safety frameworks and mechanistic understanding of in vivo efficacy, including direct head-to-head comparisons between fungal enzymes and bacterial probiotics under identical conditions. Full article

Ready-to-eat sea cucumbers (RSC) cannot be preserved at room temperature due to autolysis, which is closely related to the instability of collagen resulting from the disruption of hydrogen bonds. To investigate the protective effect of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) cross-linking against disruption of hydrogen bonds and its role in stabilizing RSC quality at room temperature, this study designed comparative experiments involving EDC/NHS cross-linking treatments with varying sequences of hydrogen bonds disruption. The results indicated that EDC/NHS positively affects the stabilization of the collagen structure in RSC. The various quality parameters of both groups of RSC that underwent cross-linking treatment before and after hydrogen bonds disruption were significantly better than those of the control group, which only experienced the breaking of hydrogen bonds. Notably, the Eb group, which underwent EDC/NHS cross-linking treatment prior to the disruption of the hydrogen bonds network, yielded even more favorable results. Preliminary analyses of textural properties and moisture content suggested that EDC/NHS helps delay the deterioration of RSC quality. The levels of soluble components and carbonyl groups indicated that prior cross-linking treatment is more effective in mitigating collagen degradation and oxidation. Differential scanning calorimetry revealed that the reduction in ΔH for the Eb group was only 2.4%. Furthermore, fluorescence spectroscopy, Fourier transform infrared spectroscopy, and circular dichroism spectroscopy, examined from the perspectives of secondary and tertiary structures respectively, indicated that the cross-linking mechanism of EDC/NHS involves the formation of a more robust network of amide bonds, thereby preventing the disruption of hydrogen bonds and enhancing collagen stability, enabling it to better resist the cleavage of hydrogen bonds due to urea. The scanning electron microscope and Van Gieson’s staining techniques offer a clearer illustration of this point from a microscopic perspective. Moreover, molecular docking simulations have indicated the cross-linking mechanism of EDC/NHS at the atomic level, thereby establishing a scientific foundation for the potential application and development of EDC/NHS in room-temperature storage technologies for RSC. Full article

Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species—Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)—are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R² ≥ 0.999) with amplification efficiencies of 90–110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens. Full article