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Hepatitis E virus (HEV) is the leading cause of hepatitis globally and poses particular risks for immunocompromised individuals. Mandatory screening of blood donations for HEV RNA and retrospective individual testing of previous donations (lookback investigations) following a reactive result have been implemented in several countries to protect these patients. This includes Germany, where a sensitivity limit of 2000 IU/mL applies to index donations. In total, 334 HEV RNA-positive blood donations were detected at our blood donation service between 2018 and 2024. Lookback testing was applied in 211 cases, revealing previous HEV RNA-positive donations in 23.1% of donors (n = 48, 76 donations). Although 16 of these retrospectively tested HEV RNA-positive donations have already been transfused, no transfusion-transmitted HEV infection has been reported. The HEV RNA viral load in the lookback donation was below 50 IU/mL in 72.4% of cases. Routine testing effectively prevents highly viremic blood products entering the supply, significantly reducing the infection risk. While the administration of virus particles with low-viremic products cannot be ruled out, the remaining risk appears to be minimal and has been deemed so far acceptable for the safety of blood products. The lookback strategy further supports the screening strategy by retrospectively identifying blood products from low-viremic donations and enabling appropriate risk management.

Over recent decades, advances in medicine have transformed the management of systemic diseases, severe inflammatory syndromes, and end-stage organ failure, expanding therapeutic possibilities in intensive care and transplantation. The fourth Workshop on Purification Therapies (WPT25), entitled "From Research to Clinic: The End of the Beginning", marked an important moment in the maturation of extracorporeal blood purification therapies (EBPTs) and organ perfusion technologies. The first day focused on dysregulated inflammatory diseases and EBPTs, highlighting the role of inflammatory mediators as cytokines, pointed out the potentiality in their clinical applications in septic and cardiogenic shock. The discussion was focused on patient selection, timing, dosing, and drug-device interactions. The second day addressed organ preservation and regeneration, emphasizing in situ and ex situ perfusion strategies to expand donor eligibility-including DCD and extended criteria donors-while mitigating the iatrogenic effects as the ischemia-reperfusion injury. Discussions explored temperature management, inflammatory modulation during procurement and treatment, and future perspectives such as personalized perfusion protocols and xenotransplantation. With 550 participants, 26 oral presentations, practical workshops, and 161 scientific contributions published in one special issue of Transplant International, the meeting consolidated evidence and try to define priorities for integrating purification and perfusion therapies into clinical practice. Abstracts from the meeting are published in Transplant International: "Abstract Book of the 4th Workshop Purification Therapies From Research to Clinics "The End of the Beginning", September 19th-20th, 2025." at https://www.frontierspartnerships.org/research-topics/197/aferetica---wpt-2025-meeting-abstract.

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we developed and validated an in-house quantitative PCR (qPCR) assay targeting ORF26, optimized for whole blood. Assay calibration used plasmid, BCBL-1 cell-derived, and commercial HHV-8 DNA standards. Analytical validation was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and showed a 95% limit of detection of 65.7 copies/reaction, efficiencies of 90-101% (R2 > 0.99), and intra/inter-assay coefficients of variation < 6.5%. Strong correlations were observed among the three calibrators (R2 > 0.97).Clinical validation against a composite reference yielded 100% sensitivity, specificity, PPV, and NPV. Viral loads (log10 copies/mL) varied by clinical condition: classic KS and transplant-associated KS showed the lowest medians (2.30-2.23), MCD HIV- and PEL intermediate values (2.83-3.72), and epidemic KS, MCD HIV+, and IRIS-KS the highest (4.12, 4.86, and 5.03, respectively). Viremia > 5 log10 copies/mL was associated with uncontrolled E-KS, MCD HIV+, and IRIS-KS. Longitudinal follow-up revealed viral load decline paralleled clinical improvement. This validated assay provides a robust, affordable tool for HHV-8 quantification in whole blood and supports its integration into diagnostic workflows and patient monitoring.

The proviral load of human T-cell lymphotropic virus type 1 (HTLV-1) is an important biomarker associated with the monitoring and risk stratification of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the lack of standardized quantification methods limits its broader application. This study evaluated a novel absolute quantification approach based on whole blood volume and compared its performance with established protocols. A total of 66 HTLV-1-infected individuals were analyzed using six qPCR-based methodologies, including volumetric quantification (copies/&#xb5;L) by absolute quantification and the Tameg&#xe3;o-Lopes method, as well as normalization per 1000 cells (whole blood, buffy coat, PBMCs, and CD4+ T cells). Association and agreement were assessed using Pearson's correlation, Bland-Altman analysis, concordance correlation coefficients (CCCs), and Deming regression. Absolute quantification showed strong correlation with both the Tameg&#xe3;o-Lopes method and CD4+-based quantification (r = 0.93 and 0.84, respectively; p < 0.001) and high agreement (CCC = 0.866 and 0.811, respectively), with modest systematic bias (-0.273 log10 copies/&#xb5;L and 0.115 log10 copies/103 cells, respectively). Leukocyte-normalized methods showed greater discrepancies, likely due to dilution by uninfected cells. These findings show that quantification based on total blood volume is a simplified, operationally feasible alternative for assessing HTLV-1 proviral load.

The detailed mechanism and sequence by which tick-borne flaviviruses (TBFVs), such as Langat virus (LGTV), infect and disseminate in arthropod hosts remain undefined. To begin characterizing these processes, we used artificial membrane feeding chambers to feed adult Ixodes scapularis ticks with blood containing LGTV. At 24, 48, 72, and 96 hours (h) after attachment, we removed and dissected the partially fed ticks to obtain the midgut and salivary glands. Histology confirmed infection in cells of the digestive epithelium lineage; infection was noted in midgut generative cells and the more differentiated functional digestive cells over the course of feeding. The viral envelope (E) protein, nonstructural protein 3 (NS3), and double-stranded RNA (dsRNA) were readily detected in these cells by 48 h after infection. Parallel analysis indicated that cells in salivary gland acini were also infected by 48 h, where virus target cells appeared to be the granular cells in acini types II and III. Thus, both salivary glands and midgut showed direct evidence of infection by 48 h. Although viral staining was not observed at 24 h, when organs were removed at 24 h and individually cultured ex vivo, the virus was detected. Taken together, our results provide evidence of LGTV infection in both the salivary glands and midgut within the first 24 h of a blood meal. The findings should prompt a reevaluation of the systemic dissemination of TBFV in infected ticks.

Population-level immune single-cell profiling has the potential to enable the association of cellular function with human ancestry, demographic, and environmental variables. However, methods for collecting and processing peripheral blood mononuclear cells (PBMCs) for single-cell sequencing are difficult to implement in remote and rural settings, resulting in a lack of representation of underserved communities across the global south. We developed SiteCELL, a method that enables purification and cryopreservation of PBMCs from whole blood at the site of collection using minimal laboratory equipment and without electricity. By comparing matched samples of purified PBMCs, we showed that SiteCELL performs as well as ficoll density gradient (FDG), both in laboratory and rural settings. This method ensures accurate recovery of cell type proportions and excels in reducing stress and minimizing variability in sampling batches across countries. Hence, SiteCELL is ideal for multinational initiatives targeting the inclusion of underrepresented ancestries in cellular atlases.

Parvovirus B19 (B19V) has been found in asymptomatic blood donors. This prospective study tested linked blood donor-recipient samples from patients transfused at the National Institutes of Health (NIH) and affiliated hospitals for the presence of B19V DNA and anti-B19 immunoglobulin G (IgG) antibodies to assess the risk of B19V transmission by transfusion. Post-transfusion recipient plasma samples collected during 2002-2020 were tested for B19V DNA at 1, 2, 4, and/or 8&#x202f;weeks by nested polymerase chain reaction (PCR), and for anti-B19 IgG at 12 and &#x2265;24&#x202f;weeks by enzyme immunoassay (EIA). To confirm B19V DNA-positive recipients, pre-transfusion and linked donation samples were tested for B19V DNA, anti-B19 IgG, and/or IgM. Tested samples included 1,352 subjects tested for DNA and anti-B19V IgG, or both. No new transfusion-transmitted B19V infections were identified. Only one confirmed transfusion-transmitted infection (TTI) was identified overall. A total of 19 of 1,314 subjects tested for B19V DNA (1.4%, 95% confidence interval [CI]: 0.014 [0.009, 0.021]) had a positive result in their post-transfusion sample. A total of 876 of 1,290 tested subjects were positive for anti-B19V IgG antibody (68%, 95% CI: 0.679 [0.65, 0.70]). Antibody detection fluctuated among some recipients from 12 to 24&#x202f;weeks. The B19V DNA-positive rate in recipients was very low. All B19V DNA-positive recipients had pre-existing antibodies, indicating prior exposure. Although these data may not provide enough evidence to consider revising current B19V testing strategies, other pooled plasma testing data from around the world suggest that more widespread B19V monitoring of blood products could be indicated to prevent B19V infection in immunosuppressed or otherwise susceptible recipients (e.g., individuals with chronic hematological diseases, pregnant women less than 20 weeks gestation).

This study aimed to investigate the distribution of pathogenic bacteria and identify clinical predictors associated with bloodstream infection (BSI) in patients with systemic lupus erythematosus (SLE). The experimental group included 43 SLE patients with BSI (SLE-BSI), who were diagnosed and treated at the First Affiliated Hospital of Sun Yat-sen University between October 2013 and June 2023. The control group consisted of 172 SLE patients without BSI (SLE-non-BSI), randomly selected at a 1:4 ratio and matched for age, gender, and ward with the case group. Logistic regression analyses of clinical data and laboratory indicators were used to identify independent clinical predictors for SLE-BSI. The most common pathogens identified in SLE-BSI patients were Staphylococcus aureus (18.6%), Salmonella spp. (16.3%) and Escherichia coli (11.6%). Multivariate logistic regression analysis revealed that fever (OR = 4.079, 95% CI: 1.582-10.517, p&#x2009;=&#x2009;0.004), myalgia (OR = 5.637, 95% CI: 1.363-23.318, p&#x2009;=&#x2009;0.017), and glucocorticoid use (OR = 2.690, 95% CI: 1.041-6.948, p&#x2009;=&#x2009;0.041) were independent clinical predictors associated with the occurrence of SLE-BSI. For SLE patients receiving glucocorticoid therapy, the presence of myalgia and fever should raise suspicion of BSI. The findings may help optimize the clinical utility of blood culture testing, thereby facilitating the early identification of BSI in SLE patients.

Cytomegalovirus (CMV) is the most common congenital infection and an important cause of childhood disability. Newborn CMV screening and studies of mother-to-child transmission are increasingly common, involving viral detection using various sample types. Here, we describe the analytical evaluation of the AltoStar&#xae; CMV PCR Kit 1.5, a commercial diagnostic platform for detection of CMV DNA in diverse sample types relevant for congenital CMV infection. Oral swabs, saliva, dried urine spots (DUS) and dried blood filter paper spots (DBS), umbilical cord blood, amniotic fluid, placental tissue, vaginal swabs, and breast milk from known CMV-negative donors were spiked with known concentrations of viral isolates. Linearity of detection in fluid samples ranged from 0.96 to 1.00. Limits of detection in fluid samples ranged from 2.22 to 2.83&#x202f;log IU/ml. CMV PCR on dried urine spot samples had greater sensitivity when using off-instrument nucleic acid purification with Quanta Extracta DBS buffer (QuantaBio). This study provides a cross-matrix performance framework for CMV molecular testing in congenital infection research and newborn screening contexts. The AltoStar&#xae; CMV PCR platform demonstrated robust performance across clinically relevant specimen types and scalable throughput capacity, supporting its suitability for large-scale epidemiologic and screening applications.

Increased expression of both the nucleosome assembly protein (NAP) superfamily member testis-specific protein Y-encoded-like 2 (TSPYL2) and N-methyl-D-aspartate receptor (NMDAR) in the hypothalamic paraventricular nucleus (PVN) is crucial for the pathogenesis of hypertension, yet how they interact during hypertension is not fully understood. It is highly possible that NMDAR can be regulated by TSPYL2 in the PVN during hypertension. To examine the hypothesis, TSPYL2 is knocked-down or overexpressed in the PVN of spontaneously hypertensive rats (SHRs) and the normotensive control Wistar-Kyoto rats (WKYs), as well as the cultured primary PVN cells of SHRs. The transcription of NMDAR subunits, together with the blood pressure, plasma norepinephrine (NE) and the inflammatory responses and oxidative stress in the PVN were examined. Results suggested that TSPYL2 knockdown reduced blood pressure, downregulated the transcription of GluN2A/B and reduced inflammatory responses and oxidative stress in the PVN; while in the PVN of WKYs, it did not cause such significant changes. In vitro examination further revealed reduced basal cellular Ca2+ concentration after TSPYL2 knockdown, indicating tampered NMDAR function. These results suggest that TSPYL2 is critical for maintaining hypertension via transcriptional regulation of GluN2A/B. Interfering with TSPYL2 can block GluN2A/B expression and suppress the function of NMDARs in the PVN, which reduces the high blood pressure of hypertensive animals.

Drug-resistant tuberculosis (DR-TB) is still a concern for public health and health security, making TB the world's largest cause of death from a single infectious agent. This Chapter was aimed at evaluating the prevalence of DR-MTB in rural communities of South Africa. A cross-sectional study in rural Vhembe District, South Africa, recruited 175 active TB patients. Data on lifestyle behaviour, socioeconomic, and environmental characteristics were collected via a questionnaire, and samples (blood and sputum) were collected. The U-rapid blood test confirmed HIV status. DNA was extracted from sputum and tested using multiplex real-time PCR assays with Anyplex and Allplex kits to detect Mycobacterium tuberculosis/nontuberculous mycobacteria, as well as DR-TB strains, respectively. The participants' ages ranged from 18 to 82&#xa0;years (mean&#xa0;=&#xa0;44&#xa0;&#xb1;&#xa0;13.0), with the majority being males (57.1%, 100/175) and unemployed (61.1%, 107/175). The co-infections detected include NTM and HIV. The prevalence of DR-MTB was 2.3% (4/175). A 20.6% (36/175) success rate was achieved in treatment outcomes. DR-TB remains low and is effectively managed, with favourable treatment outcomes reflecting effective clinical and public health interventions. However, the high prevalence of emerging NTM and HIV infections may compromise these gains, posing a potential threat to sustained TB control.

The escalating global burden of antimicrobial resistance (AMR) necessitates diagnostic strategies that can overcome the limitations of conventional culture-based methods, which often require several days to generate clinically actionable results. Such delays are associated with increased mortality, inappropriate antibiotic use, and continued transmission of resistant pathogens. In this context, microfluidic chip technology has emerged as a promising platform for rapid, miniaturized, and increasingly automated point-of-care diagnostics. Recent advances have enabled integrated lab-on-a-chip systems that combine bacterial isolation, phenotypic antimicrobial susceptibility testing, and genotypic resistance detection within closed and self-contained architectures, thereby reducing contamination risk and operator dependence. In addition, these platforms are increasingly capable of operating at single-cell resolution, allowing the detection of heteroresistance and resistant subpopulations that may be overlooked by conventional bulk assays. A major advantage of microfluidic systems is their ability to bridge phenotypic and genotypic diagnostics by enabling real-time monitoring of bacterial growth, metabolic activity, and morphological responses to antibiotics while simultaneously incorporating on-chip nucleic acid amplification for resistance gene detection. This integrated approach improves the interpretation of discrepancies between genetic determinants and functional resistance. Studies to date have demonstrated high sensitivity and specificity in complex clinical matrices, including blood, urine, and sputum, with turnaround times reduced from days to less than one hour in some applications. Furthermore, the integration of CRISPR-Cas systems, nanomaterial-enhanced biosensing, and machine learning has further improved analytical performance and data interpretation. Nevertheless, important translational challenges remain, including scalable manufacturing, regulatory standardization, and integration into routine clinical workflows. Future microfluidic platforms are expected to support multiplexed, intelligent antimicrobial susceptibility testing capable of simultaneous pathogen identification, resistance profiling, and therapeutic guidance, thereby advancing precision diagnostics for AMR management.

A previously asymptomatic male infant presented with persistent fever for 4 weeks with progressive abdominal distension due to hepatosplenomegaly. Investigations were suggestive of bicytopenia. His fever was non-responsive to intravenous antibiotics. Blood and urine cultures were sterile. Bone marrow showed normal haematopoiesis with evidence of yeast forms of Histoplasma capsulatum inside erythroblasts. Urine was positive for histoplasma antigen. The infant responded to intravenous liposomal amphotericin B. Fever resolved, anaemia and thrombocytopenia recovered. Hence, we report this case of an infant with disseminated histoplasmosis presenting with fever, hepatosplenomegaly and bicytopenia.

Crime Scene Investigators (CSIs) in the UK conduct extensive anticontamination measures to mitigate the risk of introducing extraneous DNA into forensic evidence. This study empirically evaluated the effectiveness and proportionality of such measures, focusing on equipment cleaning and glove-changing practices during evidence recovery in mock crime scenes. The experimental design deliberately introduced contamination risks, such as the use of contaminated equipment, transport containers, and gloves and included both standard and streamlined (reduced glove changes/cleaning) procedures. Six CSIs from two police forces recovered 24 biological samples (semen, blood, saliva, and touch DNA) from two mock scenes. Critically, no extraneous DNA profiles were detected in any evidential samples, even under deliberately compromised conditions or when streamlined practices halved the time required for recovery. These findings indicate that many current anticontamination measures exceed what is necessary to protect evidential integrity. The results support a more proportionate, risk-based approach, suggesting that UK police forces could safely reduce certain anticontamination practices without compromising the quality of forensic evidence. Further research in real-world settings is recommended to confirm these conclusions and guide policy.

Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium and E. faecalis, are responsible for various human infections and represent a significant global public health concern in both nosocomial and community settings. To facilitate early detection of these pathogens, we developed a rapid method for detecting E. faecium, E. faecalis, and vancomycin resistance genes (vanA and vanB) in a single reaction using multiplex recombinase polymerase amplification (M-RPA) combined with a chromatographic printed-array strip (C-PAS). A total of 106 samples, including 46 E. faecium (35 vanA-type and one vanB-type strains), 39 E. faecalis (one vanA-type and two vanB-type strains), four Enterococcus spp., and 17 non-Enterococcus spp. isolates were tested using the M-RPA-C-PAS method and compared with the conventional polymerase chain reaction (PCR) method. Specific tagged and biotinylated M-RPA primers were designed for detecting ddl (E. faecium), sodA (E. faecalis), vanA, and vanB. The M-RPA reaction was performed under isothermal conditions at 37 &#xb0;C for 20 min, and visual detection using the C-PAS for DNA signal detection within another 20 min. The performance of the M-RPA-C-PAS was initially evaluated in 12 spiked rectal swabs and 12 positive blood culture samples from patients compared with the PCR method. Compared with the PCR-based method, the M-RPA-C-PAS method demonstrated sensitivities of 95.7%, 94.9%, and 100.0% and specificities of 100.0%, 92.5%, and 100.0% for ddl, sodA, and vanA, respectively, and 93.2% specificity for vanB. The lower detection limits of the M-RPA-C-PAS method for detecting the ddl and sodA genes were 10&#x2074; and 10&#xb3; CFU/mL, respectively, whereas those of the PCR method were 10&#x2075; and 10&#x2074; CFU/mL, respectively. The lower limit of detection of both methods for vanA and vanB was 10&#x2074; CFU/mL. The M-RPA-C-PAS assay showed 100% agreement with PCR for identifying the four genes in the spiked and clinical samples. The M-RPA-C-PAS method is an alternative rapid approach with high sensitivity and specificity, demonstrating comparable performance to conventional PCR. Therefore, the M-RPA-C-PAS method represents a promising point-of-care diagnostic tool for the rapid detection of VRE, supporting timely clinical management and infection control strategies.

Hemoparasitism by T. minasense, T. devei, and T. cruzi has been reported in Callithrix jacchus and C. penicillata, which are invasive marmoset species in Rio de Janeiro. We aimed to investigate parasitism by Trypanosoma sp. in marmosets inhabiting Serop&#xe9;dica municipality in Rio de Janeiro, as part of the health monitoring efforts due to the proximity of these primates to the local population and the Universidade Federal Rural do Rio de Janeiro/UFRRJ campus. Parasite species identification was performed through microscopy combined with PCR amplification, and DNA sequencing of ca. 700&#xa0;bp fragment of the 18&#xa0;S rRNA gene. Blood samples were collected from twenty-five marmosets: twelve free-living individuals from UFRRJ campus and thirteen in captivity at CETAS-RJ. Two individuals tested positive by microscopy for Trypanosoma sp., with morphometry within the range for T. minasense. Ten individuals were positive by PCR, confirming it as a more efficient methodology for this hemoparasite detection. Sequences of the 18&#xa0;S rRNA showed similarity above 98% with T. minasense, clustering accordingly in the phylogenetic tree. Free-living marmosets presented a higher infection rate (58%) than captive animals (23%). The results suggest that captivity conditions may influence parasite transmission and fragmented landscapes maintain active trypanosomatid cycles, necessitating continuous surveillance in regions of increasing human-wildlife proximity.

BACKGROUND Capnocytophaga canimorsus is a gram-negative bacillus found in the oral cavity of dogs and cats. Human infection is uncommon but can cause severe sepsis. Although classically associated with asplenia, alcohol use disorder, or immunosuppression, a substantial proportion of cases occur in patients without identifiable risk factors. Diagnosis may be delayed due to the fastidious nature of the organism and the impact of prior antibiotic exposure on culture yield. CASE REPORT A 75-year-old woman presented with fever and right upper-quadrant abdominal pain 6 weeks after right total hip arthroplasty. She had no history of animal bite but reported close contact with dogs. Initial investigations did not identify a source of infection, and empirical ceftriaxone was initiated. Blood cultures became positive after extended incubation (Day 10), with identification of C. canimorsus by MALDI-TOF mass spectrometry. Imaging studies were inconclusive for prosthetic joint infection. The patient was treated with high-dose amoxicillin for 14 days, with favorable clinical and biological outcomes. CONCLUSIONS C. canimorsus infection should be considered in patients presenting with sepsis or persistent fever and a history of dog or cat exposure, even in the absence of a bite. Close collaboration with microbiology laboratories may be required to ensure appropriate culture conditions. Clinicians should also be aware of the potential for diagnostic uncertainty in patients with recent prosthetic implants.

Saint Kitts (St. Kitts) and Nevis is a federation of two islands located within the West Indies. St. Kitts is anecdotally known to have commercial egg production, but disease prevalence and biosecurity practices of these farms have not previously been studied. In this study, we evaluated biosecurity practices of 11 commercial farms through a biosecurity questionnaire given to the farmers and collected blood and oropharyngeal swab samples from birds at each farm to evaluate disease prevalence. All surveyed egg-layer farms had deficiencies in conceptual, structural, and procedural biosecurity. Favorably, most workers used dedicated farm shoes and clothes and limited visitors. The inadequate cleaning of houses, handling of mortality, and multi-age facilities raised concerns for disease transmission. For seroprevalence, antibodies against avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and Salmonella enterica subspecies enterica serovar Enteritidis (SE) were detected using ELISA kits. Most farms had positive antibodies to IBV (90.9%, 10/11), AIV (81.8%, 9/11), and all farms were positive for SE and NDV (100%, 11/11). Molecular testing for NDV and AIV antigen detection were negative. Chicks imported from hatcheries in other countries received vaccinations for IBV and NDV, suggesting that chicks are transmitting vaccinal strains when integrated into multi-age layer houses. SE seroprevalence in all farms, paired with poor nest availability, egg sanitation practices, and ineffective house cleaning raise food safety concerns. The results of this study highlight the need for disease prevention programs in St. Kitts commercial poultry production, to safeguard both animal and human health.

An indigenous autochthonous bacterium, Bacillus cereus PX472023 isolated from the gut of Lates calcarifer was evaluated for its potential probiotic property. The isolated bacteria produced hydrolytic enzyme activities such as starch, esculin and casein hydrolysis along with positive result for biofilm formation. The isolate possessed desirable features like tolerance to low pH (3), high bile salt (up to 4%), pepsin (0.3%), high saline conditions (up to 3%), along with optimum hydrophobicity, auto and co aggregation activity. In addition, the isolate exhibited strong antagonistic activity against L. calcarifer pathogens, including Photobacterium damselae subsp. damselae, Lactococcus garvieae, Aeromonas veronii and Pseudomonas putida. In addition, PCR amplification revealed the presence of two probiotic marker genes (LuxS gene and E1 &#x3b2;-subunit of the pyruvate dehydrogenase complex gene) as well. Draft genome assembly analyses revealed genome size to be 56,16,837&#xa0;bp, with GC content of 34.97% and 5,605 protein coding sequences. Downstream analyses following the functional annotation of the genome demonstrated the presence of variety of bacteriocin gene clusters and presence of several genes in favour of probiotic potential of the isolate. Safety evaluation demonstrated that the isolate showed no haemolysis on sheep blood agar, was devoid of virulence-associated genes in its genome and did not induce mortality in the in vivo challenge study. The genomic investigation of antimicrobial resistance determinants revealed the absence of acquired antimicrobial resistance genes in the isolate, thereby alleviating primary concerns regarding horizontal transfer of resistance genes. The integration of in vitro and genomic analyses provided preliminary evidence supporting the probiotic potential of Bacillus cereus PX472023 in L. calcarifer and highlighted its prospective suitability for in vivo application in the same host under local environmental conditions.