Search Results (633)

Severe Combined Immunodeficiency (SCID), Spinal Muscular Atrophy (SMA), and X-Linked Agammaglobulinemia (XLA) are rare but life-threatening genetic disorders in infants that can lead to severe infections, progressive neuromuscular degeneration, or severe immune dysfunction associated with significant morbidity and mortality if not diagnosed early. Advances in newborn screening (NBS) technologies have enabled pre-symptomatic detection of these conditions, allowing early initiation of life-saving interventions such as hematopoietic stem cell transplantation, gene therapy, and immunoglobulin replacement therapy. However, the absence of a standardized national clinical pathway linking screening, confirmatory testing, and specialist referral in Malaysia continues to contribute to delayed diagnosis and suboptimal patient outcomes. This review examines and synthesizes current evidence on the clinical pathways for early diagnosis and management of SCID, SMA, and XLA, with particular emphasis on diagnostic workflows, screening technologies, and healthcare system challenges within the Malaysian context. The review examines disease epidemiology, consequences of delayed diagnosis, and the role of expanded NBS under the Screening for Health, Intervention, Nurturing of Every Child (SHINE) program in improving early diagnosis and management. In addition, the paper outlines the current NBS landscape, the use of multiplex real-time polymerase chain reaction (PCR) assays for simultaneous detection of T-cell receptor excision circles (TREC), kappa-deleting recombination excision circles (KREC), and survival motor neuron 1 (SMN1) gene deletion of exon 7 from dried blood spot (DBS) samples. A structured diagnostic framework incorporating screening interpretation, confirmatory testing, and urgency-based referral pathways is also proposed. By addressing current operational barriers and coordinating laboratory referral systems, expanding NBS programs could significantly improve early diagnosis and long-term outcomes for infants affected by SCID, SMA, and XLA in Malaysia. Full article

Fucoidan, a sulfated polysaccharide, is known for its beneficial bioactive effects, for example antioxidant, anti-inflammatory, and vascular modulatory effects. Such a bioactive compound may also be useful for treating neurodegenerative diseases like age-related macular degeneration (AMD). Our research focuses on AMD-related pathomechanisms using primary porcine retinal pigment epithelium (RPE) cells in vitro and zebrafish (Danio rerio) models in vivo. We tested the bioactivity of a commercially available fucoidan (FVs) from bladderwrack with regard to pathomechanisms of AMD. We performed multiplex assays, RT-qPCR and fluorescence-based assays for the formation of nitric oxide (DAF-FM assay) and reactive oxygen species (DCF-DA assay) to analyze angiogenesis-related chemokines and pro-inflammatory cytokines as well as protection against oxidative stress and inflammatory insult. Our results showed that FVs significantly reduced the secretion of pro-angiogenic vascular endothelial growth factor A (VEGF-A) and follistatin as well as the pro-inflammatory cytokines interleukin 8 (IL-8) after lipopolysaccharide (LPS) and polyinosinic/polycytidylic acid (PIC) induction. Interleukin 6 (IL-6) was also reduced in the supernatant of the RPE cells. Additionally, in zebrafish, fucoidan decreased the production of NO and ROS. Gene expression of zebrafish embryos revealed anti-inflammatory effects by suppressing pro-inflammatory genes and significantly downregulating, e.g., interleukin 1 beta (IL-1β). These findings indicate modulation of oxidative stress, inflammatory responses, and VEGF secretion of the used FVs. This study demonstrates that fucoidan possesses AMD-relevant bioactivities in vitro and in vivo, suggesting fucoidan warrants further investigation in AMD-related research and related pathological mechanisms. Full article

Accurate detection of Campylobacter in chicken liver is hindered by strong matrix inhibition. This study evaluated sample-processing strategies to improve droplet digital PCR (ddPCR) quantification of Campylobacter coli and Campylobacter jejuni in chicken liver. Mechanical homogenization (Stomacher) and enzymatic/mechanical dissociation (gentleMACS), with and without 8 μm filtration, were compared. Particle-size analysis showed that filtration, especially following gentleMACS treatment, produced smaller, more uniform particles and reduced variability. Percent-degradation assays confirmed that gentleMACS achieved substantially greater tissue disruption than Stomacher homogenization. The multiplex ddPCR assay, which simultaneously targets C. coli and C. jejuni, produced droplet counts comparable to single-target reactions, indicating minimal interference between targets under the conditions tested. In inoculated liver samples, gentleMACS processing yielded droplet counts similar to those obtained from pure cultures, whereas unprocessed liver caused severe matrix interference and inconsistent quantification. Furthermore, gentleMACS-treated samples exhibited strong log-to-log linearity for quantifying C. coli and C. jejuni, enabling detection near 1 genome copy equivalent per reaction. Overall, the results indicate that enzymatic/mechanical dissociation combined with fine-pore filtration improves ddPCR detection of Campylobacter species in chicken liver. Full article

Escherichia coli, Clostridium perfringens and Cryptosporidium are common diarrhea-related pathogens in dairy calves, posing considerable economic losses to animal husbandry and threatening public health. A previous study in our lab found the frequent occurrence of Escherichia coli virulence genes (eaeA, stx1 and stx2), Clostridium perfringens cpa and Cryptosporidium in diarrheic dairy calves in Ningxia Hui Autonomous Region, China. The present study aimed to develop a multiplex PCR for simultaneous detection of these virulence genes and Cryptosporidium in diarrheic dairy calves. The multiplex PCR demonstrated sensitivities of 2060 copies, 18200 copies, 1300 copies, 1990 copies and 974 copies for stx1, stx2, eaeA, cpa and 18S rRNA, respectively. Moreover, the method showed no cross-reactivity with Giardia duodenalis, Enterocytozoon bieneusi, Eimeria, Haemonchus contortus, Oesophagostomum, Moniezia, Salmonella, Proteus mirabilis and Staphylococcus aureus. Further application of the multiplex PCR in 20 clinical faecal samples from diarrheic dairy calves found that the positive rates of the multiplex PCR assay were 55% (11/20), 50% (10/20), 60% (12/20), 45% (9/20) and 25% (5/20) for stx1, stx2, eaeA, cpa and 18S rRNA, respectively, which were not significantly lower than those of the conventional PCR targeting stx1 (60%, 12/20) and eaeA (65%, 13/20), but higher than those of the reported PCR targeting stx2 (45%, 9/20) and cpa (40%, 8/20), and were consistent with those of the reported nested PCR targeting 18S rRNA (25%, 5/20). Taken together, the present study preliminarily developed a multiplex PCR assay for the rapid detection of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in dairy calves, which could provide basic data and technical support for the diagnosis and prevention of calf diarrhea. However, more samples from divergent clinical settings are needed to validate the assay in the diagnosis of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in future studies. 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

Cephalopod products are widely distributed as frozen raw materials or cut portions, making morphology-based species identification difficult during commercial handling and inspection. In this study, we developed a conventional multiplex PCR assay for the simultaneous identification of six commercially important cephalopod species, Octopus vulgaris, O. ocellatus, O. minor, Enteroctopus dofleini, Dosidicus gigas, and Todarodes pacificus. Species-specific forward primers and a shared reverse primer were designed from the mitochondrial cytochrome c oxidase subunit I (COI) region to generate distinct diagnostic amplicons within a single reaction. The assay successfully produced species-resolved bands of 459, 365, 248, 194, 141, and 82 bp for O. vulgaris, E. dofleini, O. ocellatus, O. minor, D. gigas, and T. pacificus, respectively, with no ambiguous overlap among diagnostic fragments. Clear and reproducible amplification was obtained at annealing temperatures of 51–54 °C, with 52 °C selected as the standard condition, indicating useful operational tolerance for routine application. The assay also retained consistent diagnostic performance down to 1 ng of template DNA per reaction. These results demonstrate that the developed multiplex PCR assay provides a simple, rapid, and gel-based method for the preliminary identification of selected cephalopod species in frozen commercial materials and may be useful for seafood inspection and market surveillance. Full article

Background/Objectives: Arboviruses including dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), and West Nile virus (WNV) co-circulate across the Americas, generating overlapping febrile syndromes that challenge etiological diagnosis based solely on clinical criteria. Cartridge-based multiplex molecular platforms offer potential for decentralized testing in hyperendemic settings, yet independent real-world evaluations of their clinical and analytical performance remain limited. Methods: A retrospective two-phase analytical study was conducted. Phase 1 assessed clinical diagnostic accuracy for dengue using 163 de-identified serum samples classified using a composite reference standard consisting of Panbio NS1 ELISA reactivity (≥11 Panbio units) combined with compatible clinical and epidemiological data, operationalized in accordance with the PAHO 2023 laboratory confirmation algorithm for dengue; RT-qPCR was not routinely available for all archived samples, and reported sensitivity should therefore be interpreted as a conservative lower-bound estimate; Phase 2 evaluated analytical sensitivity across all eight panel targets using characterized arboviral reference strains in serial dilution experiments, with reference RT-qPCR assays as the comparator; this phase was incorporated to characterize detection thresholds for targets not represented by clinical specimens. Results: In Phase 1, the M10 demonstrated sensitivity of 96.0% (96/100), specificity of 100% (63/63), overall accuracy of 97.5%, and near-perfect agreement with the reference standard (Cohen’s κ = 0.95). DENV-3 was the predominant serotype (74/96; 77.1%), followed by DENV-1 (16.7%) and DENV-4 (6.3%); DENV-2 was not detected. In Phase 2, operational LoDs (defined as the lowest concentration yielding a detectable Ct in all triplicate reactions for the RT-qPCR, and from a single cartridge per dilution point for the STANDARD M10) were equivalent or superior to reference RT-qPCR for six targets (DENV-1, DENV-3, DENV-4, ZIKV, WNV, YFV; range 1–5 PFU/mL), while DENV-2 and CHIKV showed 20-fold higher operational LoDs (20 PFU/mL vs. 1 PFU/mL for the reference RT-qPCR); formal LoD₉₅ estimates were not determined. Conclusions: The STANDARD M10 Arbovirus Panel shows high clinical accuracy for dengue and adequate analytical sensitivity for most targets, supporting its use as a complementary decentralized molecular tool. Reduced sensitivity for DENV-2 and CHIKV and the absence of formal LoD₉₅ estimates remain key limitations to be addressed in future validation studies. Full article

Canine babesiosis caused by Babesia gibsoni is prevalent in Hong Kong. While molecular diagnosis via PCR is highly sensitive, it typically requires reference laboratory access, delaying clinical decisions. This study evaluated the diagnostic accuracy of a point-of-care multiplex PCR compared to a reference laboratory PCR for detection of B. gibsoni in dogs. A retrospective cross-sectional study was conducted on 47 canine EDTA blood samples collected between May 2024 and June 2025. Samples were tested for B. gibsoni using a point-of-care multiplex PCR analyser (POCMA) and a reference-standard real-time PCR. Clinicopathological data were evaluated when available. Diagnostic sensitivity, specificity, predictive values, and agreement between tests were calculated. The reference-standard assay detected 23 positives (49%) and 24 negatives (51%) whereas the POCMA detected 20 positives (43%) and 27 negatives (57%). The point-of-care assay demonstrated a sensitivity of 82.6% (95% CI: 62.9–93.0%) and specificity of 95.8% (95% CI: 79.8–99.3%) for B. gibsoni detection. All samples were negative for B. canis by the POCMA. The point-of-care multiplex PCR analyser (AIMDX1800) offers rapid, specific detection of B. gibsoni with excellent specificity but moderate sensitivity. Positive results in clinically compatible cases can guide prompt treatment, while negative results in symptomatic dogs in endemic areas warrant confirmation by reference laboratory PCR. The absence of B. canis detection by the POCMA is consistent with previous literature supporting its rarity in Hong Kong. Full article

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver malignancy with a rising global incidence and limited therapeutic options. Vascular invasion (VI) is a hallmark of advanced disease, correlating with early recurrence and dismal prognosis, yet its tumor microenvironment (TME) drivers remain elusive. We analyzed single-cell RNA sequencing (scRNA-seq) data from 25 ICC samples to systematically characterize the cellular composition and molecular features related to VI. By integrating bulk RNA-seq data, spatial transcriptomics, and multiplex immunofluorescence, we identified a distinct subset of tumor-like cancer-associated fibroblasts (CAFs), termed tCAFs, enriched in VI-positive tumors. Functional enrichment analyses revealed that tCAFs were prominently associated with hypoxia and angiogenesis pathways, findings corroborated by the significant upregulation of tCAF markers (MME and NT5E) in ICC-derived CAFs under hypoxic conditions in vitro. Cell–cell communication analysis and spatial mapping uncovered that tCAFs might promote VI primarily through VEGF signaling interactions with endothelial cells. Integrative bioinformatics and RT-qPCR validation identified three key functional genes in tCAFs: SLC2A1, PTGS2, and PLOD2. In endothelial sprouting assays, pharmacological inhibition of SLC2A1 exerted a pronounced suppressive effect. Consistently, sprouting assays using ICC-derived CAFs with SLC2A1 knockdown confirmed that its downregulation significantly reduced endothelial sprouting capacity. Importantly, administration of the SLC2A1 inhibitor BAY-876 effectively suppressed tumor progression and intrahepatic metastasis in the orthotopic ICC mouse model. Our findings define a VI-associated cellular ecosystem and molecular landscape in ICC, unveiling a novel hypoxia–tCAFs–endothelial cells axis. Furthermore, we identify SLC2A1 as a clinically relevant therapeutic target, offering new insights into tumor VI. Full article

Lumpy Skin Disease (LSD) is an economically significant viral disease of cattle, widely prevalent across Africa, particularly in sub-Saharan regions. In 2024, Tunisia reported its first outbreak. Understanding the genetic characteristics of lumpy skin disease virus (LSDV) and related poxviruses is critical for surveillance and control. Twenty-nine samples from 26 suspected cases were screened for LSDV using qPCR, followed by a High-Resolution Multiplex Melting (HRM) assay. Three representative samples, two LSDV-positive and one bovine papular stomatitis virus (BPSV)-positive, were subjected to whole-genome sequencing using Pacific Biosciences (PacBio) HiFi long-read technology. Phylogenetic analyses of the LSDV-marker gene RPO30 and complete genomes were performed alongside SNP and InDel profiling. The Tunisian LSDV isolates clustered with Clade 1.2.2 field strains and were 100% identical to each other and to the Italian isolate LSDV_Italy_Sardinia_2025, sharing 99.99% nucleotide identity with LSDV_V281_Nigeria. Although only two LSDV isolates were sequenced which showed no genetic differences, these findings suggest genomic stability within Clade 1.2.2. The Tunisian BPSV isolate showed high similarity (98.15–98.59%) to strains reported in Germany and Switzerland. This study presents the first genetic characterization of LSDV and BPSV in Tunisia, highlighting the importance of accurate differential diagnosis among poxviruses and continuous genomic surveillance to inform control strategies. Full article

Genetic screening of monogenic traits is important for improving genetic health and increasing favorable milk protein in dairy cattle. This study developed and applied an amplification refractory mutation system PCR (ARMS-PCR) assay to simultaneously screen 21 causal variants underlying 18 monogenic traits in Holstein cattle, including 13 recessive genetic defects, 2 milk protein loci, and 3 morphological loci. The assay was designed as a unified multiplex PCR-capillary electrophoresis workflow, enabling clear detection of allele-specific products distinguished by fragment size and fluorescent color. Sanger sequencing validation of newly incorporated loci supported the accuracy of the assay. A total of 1656 cows from 12 commercial farms were genotyped using the multiplex ARMS-PCR panel, and amplicons were analyzed by capillary electrophoresis. Carriers were detected for all genetic defects except DUMPS, with carrier frequencies ranging from 0.12% to 6.64%. The highest frequencies were observed for HH5 (6.64%) and MWS (6.58%), whereas HH3, HH1, and HCD showed intermediate frequencies of 1.81% to 3.08%; all the remaining defects were below 1%. Overall, 22.10% of sampled cows carried at least 1 defect allele, including 20.47% carrying 1 defect, 1.51% carrying 2, and 0.12% carrying 3. For milk protein loci, the desirable β-casein A2A2 and κ-casein BB genotypes occurred at frequencies of 45.83% and 14.13%, respectively, and the favorable A2A2/BB combination was present in 6.22% of sampled cows. Dominant red, recessive red, and polled alleles were rare. These results indicate that multiplex fluorescent ARMS-PCR can serve as a practical targeted screening tool for the simultaneous management of known deleterious alleles and selection of favorable monogenic variants in dairy cattle breeding. Full article

Three pathogenic species of the genus Yersinia, including Plague-associated Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, are commonly associated with human infection. Current qPCR detection methods are mainly limited to the identification of one or two Yersinia species in a single reaction tube, while multiplex assays for multiple genera have been more commonly reported. Therefore, the present study aimed to establish a multiplex TaqMan qPCR assay for the simultaneous detection of these three pathogenic Yersinia species. Primer and probe sets were designed based on the inv gene for Y. pseudotuberculosis, the caf1 gene for Y. pestis, and the foxA gene for Y. enterocolitica. Under the optimized reaction conditions, the standard curve slopes for the caf1, inv, and foxA genes were −3.046, −2.968, and −2.948, respectively. The correlation coefficients (R²) ranged from 0.993 to 0.996, while the amplification efficiencies ranged from 109% to 115%. The limits of detection (LOD) were determined to be 5 × 10² copies/μL for inv (FAM), 1 × 10¹ copies/μL for caf1 (ROX), and 1 × 10¹ copies/μL for foxA (CY5). The sensitivity of the multiplex qPCR assay was 10- to 100-fold higher than that of conventional PCR, depending on the target. Specificity experiments demonstrated that no cross-reactivity was observed with non-target bacteria, including Francisella tularensis, Brucella spp., Vibrio cholerae, Salmonella Typhi, and Shigella spp. The intra-assay coefficients of variation (CVs) ranged from 0.13% to 0.79%, whereas the inter-assay CVs ranged from 0.62% to 2.61%. Among 173 spleen samples collected from wild rodents, no positive signal for Y. pestis or Y. pseudotuberculosis was detected. In contrast, Y. enterocolitica was detected in three samples (1.73%, 3/173). In conclusion, the multiplex qPCR assay developed in this study provides a sensitive and specific tool for the simultaneous detection of three pathogenic Yersinia species and has the potential to improve detection efficiency in clinical and epidemiological investigations. Full article

Brucellosis remains a significant zoonotic disease globally, causing considerable economic and public health consequences, particularly in high-risk areas of South Africa (SA) with extensive animal production, including the Limpopo and Free State provinces. This study used a combination of serological, microbiological, and molecular assays to investigate the prevalence and characteristics of Brucella in 580 slaughtered livestock (384 cattle and 196 sheep) in Limpopo and the Free State (192 cattle and 98 sheep in each province) and to compare the diagnostic findings obtained using these assays. Tissue samples collected from each animal included liver, lung, spleen, and lymph nodes. Using the standard diagnostic rose Bengal test (RBT) and complement fixation test (CFT) in series (RBT-CFT), only 2.6% (10/384) of cattle were positive, with 2.1% (95% CI: 0.7–4.9) and 3.1% (95% CI: 1.3–6.3) in the Free State and Limpopo, respectively, while 4.7% (18/384) were positive by RBT-indirect enzyme-linked immunosorbent assay (iELISA), with 5.2% (95% CI: 2.7–9.0) and 4.2% (95% CI: 2.0–7.7) in the Free State and Limpopo, respectively. The molecular prevalence was 18.5% for Brucella deoxyribonucleic acid (DNA), while 97% of PCR-positive cattle tested seronegative in both provinces. Ovine samples were largely seronegative despite showing higher polymerase chain reaction (PCR) positivity (28.6%). Brucella abortus predominated in both species, with B. melitensis and occasional mixed infections also detected. Mixed infections in this study were defined as samples producing multiple species-specific bands within the same sample, consistent with the presence of more than one Brucella species during multiplex PCR analysis. While liver and spleen tissue provided reliable PCR detection, bacterial culture yielded a 0% isolation rate. These findings, together with reports from studies in brucellosis-endemic areas in sub-Saharan Africa, demonstrate discrepancies between traditional serological methods and molecular methods, including detection of Brucella DNA in seronegative animals. This may indicate that some infected animals (chronic or latent carriers) are not identified by conventional serological assays alone, particularly in endemic settings. To strengthen surveillance and enhance the detection of exposed animals, the incorporation of iELISA as a complementary tool into South Africa’s routine surveillance programmes may be beneficial, particularly when combined with regular testing of all animals. Full article

Lactobacillus species are widely used in various food products, including conventional food products, dairy products, and health food products. To achieve the desired functional properties, manufacturers commonly incorporate two or more distinct Lactobacillus species during production. In this study, a multiplex PCR detection method was developed for four Lactobacillus species commonly used in food based on TaqMan real-time fluorescent PCR technology, enabling the efficient and rapid identification of multiple Lactobacillus strains in food matrices. The research team selected and validated four representative species—Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus paracasei—as targets for the multiplex PCR assay, designing specific primer–probe combinations for each. The accuracy and reliability of the detection method were rigorously evaluated through a series of validation experiments, including the assessment of primer–probe specificity, optimization of fluorescent signal labeling chemistries, determination of the limits of detection for individual strains, evaluation of the method’s repeatability, and analysis of commercial food samples. The results demonstrated that the selected primer–probe sets exhibited no cross-reactivity in the multiplex system and specifically amplified their target Lactobacillus species, with no amplification observed for non-target strains. The established method achieved a minimum LOD for L. acidophilus of 10² CFU/g and showed high repeatability across replicates. Furthermore, the successful detection of labeled Lactobacillus strains in commercial products confirmed the method’s practical applicability. Therefore, the developed multiplex real-time PCR assay provides a reliable, sensitive, and high-throughput tool for the simultaneous detection of multiple Lactobacillus species in complex food products and holds potential for application in quality control, product authentication, and regulatory compliance monitoring. Full article

Staphylococcus aureus nasal carriage contributes to asymptomatic transmission in both community and healthcare settings. This study aimed to characterize S. aureus strains isolated from students of the Pomeranian Medical University in Szczecin, Poland, using phenotypic and genotypic methods. A total of 175 S. aureus strains were isolated from the nasal vestibules of 800 students between 2014 and 2015. Species identification and antimicrobial susceptibility testing were performed using standard microbiological methods, while virulence-associated genes and agr groups were analyzed using Single-PCR and Multiplex-PCR assays. Genotypic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The prevalence of S. aureus nasal carriage among students was 21.9% and did not differ according to faculty or year of study. Most isolates (84.0%) were susceptible to all tested antibiotics, and no methicillin-resistant S. aureus (MRSA) strains were detected. All strains carried the hla gene, whereas hld and hlg were identified in 93.7% and 93.1% of isolates, respectively. In addition, the tst gene was detected in 22.3% of strains, while the lukS-PV/lukF-PV genes were identified in only one isolate (0.6%). The most prevalent enterotoxin genes were sep (17.1%) and sea (13.7%), whereas genes of the egc cluster, including seg, sei, and seo, were detected in 53.7% of isolates. Significant associations were observed between specific egc gene combinations and superantigen gene profiles, including increased frequencies of sec, sel, and tst genes (p < 0.001). The predominant agr type was agr-1 (49.7%), followed by agr-3 (28.6%) and agr-2 (20.0%). Strains carrying agr-1 more frequently harbored the g i m n o cluster as well as the sec, sel, and sep genes, whereas agr-3-positive isolates were significantly associated with the g i m o u and g i o u clusters and with the presence of tst, sea, and seh genes (p < 0.05). PFGE analysis demonstrated substantial genetic heterogeneity among the isolates, with no evidence of a predominant clonal lineage. These findings indicate a heterogeneous, non-epidemic population structure of S. aureus strains circulating among university students and highlight the considerable diversity and interrelationships of virulence-associated genetic profiles within this population. Full article