PURPOSE:Among various carbapenemase-producing Enterobacterales (CPE), strains producing metallo-β-lactamases (MBLs)-particularly New Delhi MBL (NDM)-exhibit the most severe resistant phenotypes. This study aimed to identify effective antibiotics or non-antibiotic agents against MBL-producing Enterobacterales. METHODS:An extensive literature search of Google Scholar and the PubMed database was conducted for articles published during 2010-2025 using the search terms (novel antibiotics OR phage) AND (MBL) AND (Enterobacterales OR Enterobacteriaceae). RESULTS:Cefiderocol exhibited lower susceptibility rates compared to aztreonam-avibactam against NDM-producing Enterobacterales isolates according to the 2025 European Committee on Antimicrobial Susceptibility Testing guidelines. Among novel β-lactamase inhibitors, taniborbactam (TAN), xeruborbactam (XER), zidebactam (ZID), and ANT2681 have demonstrated activity against MBLs. Nacubactam (NAC), when combined with partner β-lactams, is active in vitro against CPE isolates harboring blaNDM genes. TAN, XER, ZID, NAC, and ANT2681 are being developed in combination with meropenem, or cefepime for the treatment of infections caused by CPE, including various MBL producers. Additionally, due to the availability of both oral and parenteral formulations, regimens such as XER paired with ceftibuten or tebipenem are currently under evaluation for the treatment of infections caused by serine carbapenemase- and MBL-producing Enterobacterales. Moreover, the novel monobactam LYS228 alone has also demonstrated good in vitro potency against MBL producers. Novel polymyxin derivatives-including SPR741 (combined with mecillinam), SPR206, and QPX9003-and bacteriophages have shown promising activity against MBL-producing Enterobacterales. CONCLUSIONS:Continued monitoring of resistance trends and the development of innovative therapeutic strategies is warranted in the ongoing battle against CPE harboring blaMBL.

We evaluated cefiderocol activity against carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Acinetobacter baumannii (CRAB), characterized the β-lactamase gene profiles of cefiderocol-nonsusceptible CRPA by metallo-β-lactamase (MBL) subtype, and assessed cross-resistance with comparator agents. Cefiderocol minimum inhibitory concentrations (MICs) for 890 CRPA and 1,063 CRAB isolates from the 2024 Antimicrobial Testing Leadership and Surveillance (ATLAS) program were determined by broth microdilution using iron-depleted cation-adjusted Mueller-Hinton broth. Cefiderocol nonsusceptibility was defined as MIC ≥ 8 mg/L per CLSI (intermediate + resistant). DTRPA was defined as nonsusceptible to all anti-pseudomonal β-lactams and fluoroquinolones; DTRAB was defined as nonsusceptible to carbapenems, ampicillin-sulbactam, and fluoroquinolones, reflecting species-specific susceptibility panels. CRPA isolates underwent molecular screening for blaNDM, blaVIM, blaIMP, serine carbapenemase, and ESBL genes; genotype data were not available for A. baumannii. Cefiderocol susceptibility was 93.9% for CRPA and 88.5% for CRAB, respectively (nonsusceptibility 6.1% and 11.5%, respectively; OR = 2.01, P < 0.001); nonsusceptibility among DTRPA and DTRAB was 8.1% and 11.7%, respectively. Among 54 cefiderocol-nonsusceptible CRPA, 41 (76%) harbored no detectable carbapenemase gene. Nonsusceptibility among MBL-positive CRPA was exclusively attributable to blaNDM producers (27.7%, 13/47); blaVIM-positive (n = 68) and blaIMP-positive (n = 21) isolates were uniformly susceptible. Among cefiderocol-nonsusceptible CRPA, 98.1% were co-resistant to ceftazidime-avibactam and 96.3% to ceftolozane-tazobactam.

Background: Respiratory syncytial virus (RSV) is a leading cause of respiratory illness in young children. The COVID-19 pandemic profoundly altered RSV circulation, yet the regional molecular and structural characteristics of circulating RSV strains in Taiwan during this period remain incompletely characterized. Methods: We conducted a retrospective study of RSV infections identified by viral culture at a tertiary medical center in Central Taiwan from 2016 to 2023. Epidemiological patterns, clinical features, and outcomes were analyzed. A representative subset of culture-positive isolates underwent F and G gene sequencing, phylogenetic analysis, and AlphaFold2-based structural modeling to examine lineage-specific substitutions in the RSV F protein and their spatial relationship to known antigenic sites. Results: Among 277 RSV cases, seasonality was markedly disrupted during and after the COVID-19 pandemic, with delayed and altered epidemic peaks. Children aged 1–4 years accounted for the largest proportion of cases (44.6%). Of 25 sequenced isolates, 16 were RSV-A and 9 were RSV-B. RSV-A clustered within the ON1 lineage, whereas RSV-B clustered within the GB5 lineage, consistent with contemporaneous regional surveillance. RSV-B isolates demonstrated slower in vitro replication than RSV-A isolates (8.4 vs. 5.4 days). RSV-A isolates exhibited conserved F protein substitutions, whereas RSV-B isolates showed greater sequence heterogeneity. Structural modeling revealed preservation of the prefusion F protein architecture, with substitutions localized to epitope and non-epitope regions but not overlapping known monoclonal antibody resistance sites. Conclusions: RSV epidemiology in Central Taiwan was substantially altered during the COVID-19 era, including a transient RSV-B–dominant period in 2022. Although limited by sample size, integration of genomic and structural analyses provides mechanistic context for regional RSV evolution and complements larger surveillance studies. Continued regional monitoring incorporating structural approaches may inform RSV vaccine and therapeutic strategies.

We integrated genetic and phenotypic analyses to characterize carbapenem-resistant hypervirulent K. pneumoniae (CRhvKp) and liver abscess-associated K. pneumoniae (LAKp) isolates, aiming to identify potential biomarkers for the accurate detection of hvKp. A total of 660 carbapenem-resistant K. pneumoniae (CRKp) and 69 LAKp isolates were screened for five hvKp-associated biomarkers (peg-344, iroB, iucA, prmpA, prmpA2) and the string test. Among LAKp isolates, 49.3% were positive for all five biomarkers and the string test (5 genes-string test positive), while 39.1% carried all markers but were string test-negative (5 genes-string test negative). The seven-day post-infection survival rates of larvae infected with 5 genes-string test positive, 5 genes-string test negative, and other LAKp isolates were 5.9%, 10.4%, and 4.3%, respectively. In contrast, larvae infected with CRKp isolates showed higher survival rates of 7.1%, 17.9%, and 37.5% for the other groups. NTUH-K2044 and the hypervirulent isolates CRKp3, CRKp117, and LAKp88 exhibited higher lethality in bacteremia mice model compared to CRKp isolates lacking five virulence genes and string test negative. Moreover, 5 genes-string test negative LAKp produced the highest levels of capsule (117.7 ng/10⁶ CFU), followed by 5 genes-string test positive LAKp (101.4 ng/10⁶ CFU). Notably, carbapenem-resistant classical K. pneumoniae (CRcKp) isolates lacking five virulence genes and string test negative, markedly lower capsule levels (7.4 ng/10⁶ CFU) compared to other groups. These results suggest that the combined use of the five genetic markers and the string test enables effective identification of hvKp, and the capsule production is correlated with the virulence potential of the strains.IMPORTANCEThis study tackles the growing threat of K. pneumoniae strains that combine high antibiotic resistance with hypervirulence. By integrating genetic and phenotypic markers, we examined carbapenem-resistant K. pneumoniae (CRKp) and liver abscess-associated K. pneumoniae (LAKp) isolates. We found strong associations between five key virulence genes (peg-344, iroB, iucA, prmpA, prmpA2), positive string test results, high capsule production, and tellurite resistance. A 23-year surveillance of 1,815 carbapenem-susceptible isolates revealed that the proportion of blood isolates carrying all five virulence biomarkers, regardless of string test positivity, was significantly higher than that of urine isolates. Our findings provide a practical framework to enhance detection and risk assessment of hypervirulent K. pneumoniae, while deepening understanding of the traits that drive their clinical impact.

BACKGROUND:Long-term care facilities (LTCFs) are highly vulnerable to healthcare-associated infections, yet evidence evaluating policy-driven, incentive-based infection prevention and control (IPC) programs in these settings remains limited. METHODS:We conducted a retrospective policy evaluation using routinely collected on-site audit and administrative data from 59 LTCFs participating in the "2025 Taichung City On-site Inspection Program for Incentives to Strengthen Infection Control in Long-term Care Facilities". Facilities were assessed using standardized IPC indicators, including mandatory core indicators (Indicators 1-3) and voluntary incentive-based indicators (Indicators 4-6). Outcomes included indicator-level compliance, incentive qualification, reward distribution by facility type and bed-capacity category, and the results of post-audit appeals. RESULTS:Compliance with mandatory core IPC indicators was consistently high: all facilities met Indicator 1 (100%), and 58 facilities complied with Indicators 2 and 3 (both 98.3%). In contrast, non-compliance was more frequently observed among incentive-based indicators. Although 34 (57.6%) facilities applied for Indicator 4 and 48 (81.4%) for Indicator 5, non-compliance occurred in 8.8% and 4.2% of applicants, respectively. Indicator 6 demonstrated the greatest implementation gap, with 13 (76.5%) of 17 applicant facilities classified as non-compliant, primarily due to ineligibility of certified personnel. All nine appealed audit items were approved after review. Overall, 58 facilities (98.3%) qualified for incentive payments. CONCLUSIONS:A government-led, incentive-based IPC audit program was associated with near-universal compliance with core IPC standards in LTCFs. Linking structured audits, preparedness requirements, and financial incentives represents a feasible approach to strengthening IPC capacity in LTCFs.

Maraviroc (MVC), a CCR5 antagonist, has been proposed as a potential antiviral agent against SARS-CoV-2; however, its mechanism of action across viral variants remains unclear. Here, we evaluated the antiviral activity of MVC against SARS-CoV-2 wild-type (WT) and Omicron BA.1 variants using single-round infectious particles (SRIPs), virus-like particles (VLPs), and cell-based assays, with a focus on its impact on viral entry and Mpro function. MVC potently inhibited infection of both WT and BA.1 SRIPs in Vero E6 cells, exhibiting EC50 values of 0.0065 μM and 0.016 μM, respectively. Time-of-addition assays revealed that MVC primarily targets the early phase of infection, with the strongest inhibition observed at the viral entry stage, while moderate effects were detected during attachment and post-entry stages. Fluorescence-labeled VLP imaging demonstrated distinct entry pathways, with WT predominantly entering via plasma membrane fusion and BA.1 via endocytosis, independent of cell type. MVC altered WT-VLP trafficking by promoting internalization and lysosomal localization, whereas it had minimal impact on BA.1 internalization. In spike-mediated cell–cell fusion assays, MVC preferentially inhibited WT spike-driven syncytium formation but showed limited effects on BA.1 or BA.4 fusion, while more effectively reducing Omicron spike-mediated binding. At the post-entry stage, MVC inhibited SARS-CoV-2 main protease (Mpro) activity, with BA.1 Mpro (P132H) exhibiting greater sensitivity (IC50 = 0.496 µM) than WT (1.869 µM). Collectively, these findings demonstrate that MVC exerts variant-dependent antiviral effects by targeting viral entry, modulating trafficking pathways, and inhibiting Mpro activity. This study highlights MVC as a multi-stage inhibitor with differential efficacy against SARS-CoV-2 variants, providing insights into its potential therapeutic application.

BACKGROUND:Fragmented and siloed nature of healthcare data leads to insufficient recognition of infection control priorities and poor adherence to preventive measures. We evaluated the impact of a visualized and integrated online information system on the infection control practice and incidence of multidrug-resistant organisms (MDROs), antimicrobial consumptions, and clinical outcomes in a medical intensive care unit (ICU). METHODS:Patients hospitalized in one medical ICU during July 1, 2023, to November 30, 2024, were collected, and were analyzed by clinical outcomes, the incidence rate of healthcare-associated MDROs, and the antimicrobial consumptions before and after the implementation of Infection Control Map (ICM). Another ICU was selected as comparison. RESULTS:A total of 775 patients were included and divided into two periods: Pre-intervention period (n = 262), and Intervention period (n = 513). There were no statistical differences among demographics except the Acute Physiologic Assessment and Chronic Health Evaluation II (APACHE II) score, which was higher in the Intervention period than that of Pre-intervention period (30.0 vs 25.0, p = 0.000). The incidence rate of MDROs decreased over time (tau = -0.53, p = 0.019), especially the methicillin-resistant Staphylococcus aureus (1.5 vs 0.3 per 1000 patient-days, incidence rate ratio = 0.2, p = 0.012), as well as the consumption of meropenem (tau = -0.53, p = 0.003). There was significant decrease in 30-day all-cause mortality after the ICM use by multivariate analysis. CONCLUSION:The implementation of the ICM significantly strengthened infection control practices, resulting in a marked reduction in healthcare-associated MRSA incidence, targeted antimicrobial usage, and 30-day all-cause mortality.

OBJECTIVES:To evaluate cefiderocol (FDC) activity against carbapenem-resistant Enterobacterales (CRE) globally, characterize the β-lactamase gene profiles of non-susceptible isolates by metallo-β-lactamase status, and assess alternative agents. METHODS:Cefiderocol minimum inhibitory concentrations for 1657 CRE isolates from the 2024 Antimicrobial Testing Leadership and Surveillance programme were determined by broth microdilution. Isolates underwent molecular screening for carbapenemase, extended-spectrum β-lactamase, and AmpC genes. Between-group differences were assessed using odds ratios (ORs) with Fisher's exact tests. RESULTS:Overall FDC susceptibility rate was 93.1% by Clinical and Laboratory Standards Institute and 80.7% by EUCAST criteria, respectively. Non-susceptibility varied substantially by genus: Escherichia spp. 25.3%, Providencia spp. 14.0%, Enterobacter spp. 12.5%, and Klebsiella spp. 3.5%. FDC non-susceptibility rates revealed further heterogeneity within genera; notably, Enterobacter cloacae (23.1%) and Enterobacter kobei (50.0%) had substantially higher non-susceptibility than Enterobacter hormaechei (7.2%), and Providencia rettgeri (22.2%) exceeded Providencia stuartii (8.0%). Asia had the highest unadjusted non-susceptibility rate (10.5%; OR 4.73, P = 0.002); however, in a multivariable logistic regression adjusting for genus, age, and specimen source, genus was the dominant independent predictor (Escherichia spp. adjusted OR 8.21, 95% confidence interval 4.75-14.17), and the Asia effect was attenuated to a non-significant trend (adjusted OR 3.99, 0.93-17.14, P = 0.063). Among 115 non-susceptible isolates, 51 (44.3%) were metallo-β-lactamase-positive, predominantly blaNDM (94.1%), with high extended-spectrum β-lactamase co-carriage (blaCTX-M 74.5%), while 50 (43.5%) harboured no identifiable carbapenemase gene. CONCLUSIONS:FDC remains active against most CRE globally, although non-susceptibility varies by genus, species and region. Notably, nearly half of non-susceptible isolates lacked detectable carbapenemase genes.

Deep vein thrombosis (DVT) remains difficult to distinguish because of its often silent presentation and the limited specificity of current diagnostic tools. We aimed to evaluate whether integrating gut microbiome profiles with routine clinical data could enhance the classification performance for identifying DVT in a case-control cohort. Stool samples were collected from individuals with DVT (n = 58), coronary artery disease (CAD, n = 56), and healthy controls (HC, n = 500). Full-length 16S rRNA gene sequencing was used to characterize the gut microbiota at species-level resolution. A random forest classifier was trained using a nested cross-validation framework, with permutation importance and SHAP (Shapley additive explanations) analyses applied to assess model interpretability. Decision curve analysis (DCA) was employed to evaluate the discriminative value of the models in an independent test set. Following linear discriminant analysis (LDA) effect size (LEfSe) screening, 95 candidate microbial features were entered into a random forest framework. Features were reduced using mutual information filtering and embedded selection to retain the final 10 for DVT vs. non-DVT classification. The integrated microbial-clinical model demonstrated substantially improved discrimination compared with the clinical-only model, achieving higher ROC-AUC [0.947 (95% CI 0.870-0.991) vs. 0.874 (95% CI 0.794-0.941)] and PR-AUC [0.793 (95% CI 0.602-0.931) vs. 0.497 (95% CI 0.274-0.724)]. Importantly, the microbiome-derived signals were robustly associated with DVT risk after adjustment for clinical covariates. Functional prediction analysis indicated enrichment of vitamin K2 and lipopolysaccharide (LPS) biosynthesis pathways in DVT, suggesting potential microbial links to coagulation and inflammation, whereas healthy controls were predominantly enriched in NAD and tetrahydrofolate (THF) biosynthesis pathways. Together, these results demonstrate that microbiome-based classification provides complementary biological insights that distinguish DVT cases from controls.

Gout is influenced by genetic and lifestyle factors, with ABCG2 variants (rs2231142 Q141K and rs72552713 Q126X) implicated in urate transport and susceptibility. Standard statistical models often yield biased estimates in rare-event cohorts. This cross-sectional study (N = 324, 15 gout cases) used Firth-corrected logistic regression to mitigate small-sample bias and address complete separation in ABCG2 genotyping data. Model performance was evaluated via internal validation with 1000 bootstrap iterations. Q126X was nearly monomorphic (T allele 0.15%), while Q141K showed higher diversity (A allele 28%). Male gender was the strongest predictor (Firth OR 8.1-11.9, P < 0.001), followed by ABCG2 dysfunction (dose-dependent risk, P = 0.055 for severe dysfunction) and alcohol consumption (Firth OR 5.26 for infrequent drinking, P = 0.022). Firth regression successfully corrected upward ML bias (e.g., alcohol OR: 8.30→5.26). The integrated model achieved an apparent AUC of 0.857 and an optimism-corrected AUC of 0.818 via bootstrap validation, demonstrating synergistic effects of genetic, demographic, and lifestyle factors. Robustness was further confirmed by high E-values (e.g., 9.99 for alcohol). These findings illustrate the utility of Firth regression for bias-reduced risk quantification in rare-event studies.

Invasive fungal endocarditis is a rare but highly lethal infection, and diagnostic accuracy is often hindered by misidentification of uncommon yeasts. Kodamaea ohmeri, in particular, is frequently mistaken for Candida species by conventional microbiological methods, leading to delays in definitive diagnosis and appropriate therapy. We report a 58-year-old male with a history of intravenous drug use who presented with acute right lower limb ischemia from femoral artery thrombosis. Despite emergent thrombectomy, persistent fever developed, and blood cultures yielded yeast that was ultimately identified as K. ohmeri by internal transcribed spacer and 26S rRNA gene sequencing. Transthoracic echocardiography revealed a large mitral valve vegetation, and fungal elements were identified within the arterial thrombus. Antifungal therapy with amphotericin B, followed by a liposomal formulation, successfully cleared fungemia. Severe valvular destruction necessitated mitral valve replacement, and molecular testing of excised tissue confirmed K. ohmeri infection. The patient completed prolonged antifungal therapy with oral voriconazole suppression and remained recurrence-free at 2-year follow-up. This case underscores the diagnostic challenges posed by rare yeasts and highlights the importance of early molecular identification, targeted antifungal therapy, and timely surgical intervention to optimize outcomes in K. ohmeri endocarditis.

OBJECTIVES:Aztreonam-avibactam (ATM-AVI) and cefiderocol (CFDC) are two next-generation antibiotics that exhibit promising in vitro activity against metallo-β-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE). However, differences in antibacterial spectra and resistance mechanisms among clinically important Gram-negative bacteria (GNB) between these two agents remain incompletely understood. METHODS:English-language publications indexed in PubMed (1990-2026), records retrieved from Google Scholar, and the 2018-2024 Antimicrobial Testing Leadership and Surveillance database were extensively reviewed to assess their potential clinical utility. RESULTS:ATM-AVI demonstrates broad activity against KPC-, MBL- and OXA-48/181-like-producing CRE. However, ATM-AVI non-susceptibility occurs in Escherichia coli and Providencia rettgeri isolates harboring blaCMY, penicillin-binding protein 3 (PBP3) modifications, and porin dysfunction or acrA efflux overexpression. In contrast, CFDC activity is primarily compromised by NDM production, reduced expression of iron transporters, and PBP3 alterations. Significant regional variation in CFDC susceptibility among MBL-producing CRE isolates collected in Asia and North America/Europe has been observed. Based on limited pooled real-world treatment experience, CFDC shows moderate efficacy (73%) for the treatment of infections caused by MBL-producing CRE. Both antibiotics also show potential in vitro activity against ceftazidime-avibactam-resistant, non-carbapenemase-producing CRE. Notably, CFDC exhibits excellent in vitro activity against contemporary CR-Pseudomonas aeruginosa, most CR-Acinetobacter baumannii harboring blaOXA-23/24/58 genes, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Burkholderia cepacia and Burkholderia pseudomallei. Nevertheless, CFDC heteroresistance is most prevalent among CR-A. baumannii isolates. CONCLUSIONS:Continued assessment of clinical treatment experience with these two antibiotics against MBL-producing CRE and important non-fermenting GNB is warranted to accumulate further evidence regarding their therapeutic roles.

Carbapenem-resistant Enterobacterales (CRE) pose significant treatment challenges. While ceftazidime-avibactam (CZA) is commonly used, resistance rates have been increasing. Aztreonam-avibactam (ATM-AVI) may represent a promising alternative. A total of 109,603 Enterobacterales isolates were collected from 307 sites across 63 countries between 2019 and 2023 as part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) program. CRE were defined as isolates with a meropenem minimal inhibitory concentration (MIC) ≥2 mg/L. Susceptibility testing was conducted according to the Clinical and Laboratory Standards Institute (CLSI) 2025 guidelines, and β-lactamase genes were identified by multiplex PCR and sequencing. Of the total Enterobacterales isolates, 7,520 (7.6%) were identified as CRE, with Klebsiella species accounting for the majority (76.3%, 5,735/7,520). CZA susceptibility was 49.1% (3,696/7,520), with a significant increase in resistance from 42.1% (540/1,283) in 2019 to 61.0% (875/1,435) in 2023 (P < 0.05). Although CRE prevalence was highest in Asia (12.2%, 2,850/23,295), the highest rate of CZA resistance was observed in Africa/Middle East (73.9%, 557/754). In contrast, ATM-AVI demonstrated the highest in vitro activity, with 97.4% (7,324/7,520) of CRE isolates exhibiting MICs ≤4 mg/L. This activity remained strong against carbapenemase-producing strains, including metallo-β-lactamase (MBL) producers. However, reduced susceptibility was observed in Escherichia (80.2%, 556/693) and Proteus (88.9%, 56/63) species. Notably, resistance to ATM-AVI among carbapenem-resistant Escherichia species was geographically clustered in India, where 9.9% (36/364) of isolates were resistant. In conclusion, ATM-AVI exhibits potent activity against global CRE, including MBL producers, and outperforms other β-lactam/β-lactamase inhibitor combinations. However, emerging resistance in Escherichia and Proteus species-particularly with regional clustering-highlights the importance of continued global surveillance.

Targeted next-generation sequencing (tNGS) has demonstrated higher analytical detection of respiratory pathogens than conventional microbiological tests (CMTs), yet its real-world clinical value in intensive care units (ICUs) remains uncertain. We conducted an interim analysis of a prospective study including 51 adult ICU patients with severe pneumonia at a tertiary medical center in Taiwan and examined the challenges to its routine implementation in the ICU. Bronchoalveolar lavage samples were analyzed using a hybrid capture–based Respiratory Pathogen ID/AMR Enrichment Panel (RPIP) and compared with standard testing (CMT combined with the FilmArray Pneumonia Panel [FAPP]). Causative pathogens were determined through multidisciplinary expert adjudication. RPIP showed a higher analytical detection rate than standard testing (90.2

A 46-year-old Taiwanese man returning from Indonesia presented with fever and thrombocytopenia. An automated cellular morphology analyzer flagged artefactual cells without a definitive diagnosis. Peripheral blood smear and PCR confirmed Plasmodium vivax. He was treated with artemether-lumefantrine and primaquine, with rapid recovery and no relapse.

The detection of neutralizing antibodies (NAbs) is crucial for evaluating immune protection against viral infections. This study assessed two ELISA-based surrogate virus neutralization tests (sVNTs): the novel GeneTex U-OK SARS-CoV-2 Neutralizing Antibody Detection ELISA kit and the FDA-authorized GenScript cPass SARS-CoV-2 Neutralization Antibody Detection kit. The GeneTex U-OK kit contains recombinant spike proteins from the wild-type strain and Omicron subvariants BA.1, BA.2, and BA.4/BA.5. A total of 115 serum samples were analyzed, including 75 from SARS-CoV-2 unvaccinated individuals and 40 from vaccinated individuals. No participant had evidence of prior SARS-CoV-2 infection, as confirmed by negative anti-nucleocapsid antibody tests. Compared with the GenScript cPass kit, the GeneTex U-OK kit demonstrated 100% positive agreement (95% CI: 90.4-100.0%) and 100% negative agreement (95% CI: 95.4-100.0%) in detecting NAbs against the wild-type strain, corresponding to 100% sensitivity and 100% specificity. When compared with viral neutralization assays, it achieved 100% concordance for Omicron BA.1, BA.2, and BA.4/BA.5 subvariants, with correlation coefficients (R) of 0.85, 0.98, and 0.46, respectively. These findings highlight the accuracy and reliability of the GeneTex U-OK kit, indicating its potential as a valuable tool for monitoring vaccine effectiveness and identifying individuals with protective NAbs against vaccinated individuals, including Omicron variants.

The BioFire Joint Infection (JI) Panel is a multiplex polymerase chain reaction assay developed for rapid pathogen detection in synovial fluid, particularly for suspected prosthetic joint infections (PJI). However, its diagnostic sensitivity varies by clinical context and pathogen, and the impact of prior antibiotic exposure on sample quality and PJI stewardship remains unclear. This prospective study evaluated the diagnostic performance of the Investigational Use Only JI Panel versus conventional synovial fluid culture, using Musculoskeletal Infection Society criteria and symptom onset within 3 weeks to define acute PJI. Fifty-four fresh synovial fluid samples from patients with suspected knee PJI were analyzed. In acute PJI, the JI Panel demonstrated comparable sensitivity to synovial fluid culture (80% vs. 95%; P = 0.096), while significantly reducing time to pathogen identification (mean 18.2 vs. 84.6 hours, P < 0.001), enabling earlier targeted antibiotic therapy in 83.3% of cases. Overall sensitivity across all PJI cases was moderate (50%) and significantly lower in chronic PJI (16.7%), reflecting limited panel coverage and reduced detection in prolonged infections. Prior antibiotic exposure reduced sensitivity for both methods, though not significant (P = 0.127). The absence of key pathogens such as Staphylococcus epidermidis and Cutibacterium acnes further limited diagnostic yield in chronic cases. The JI Panel offers a rapid and clinically impactful tool for diagnosing acute PJI and guiding antibiotic stewardship. However, its limitations in chronic PJI and susceptibility to antibiotic pretreatment restrict its standalone diagnostic utility. Therefore, it should complement, not replace, traditional culture methods, particularly in chronic PJI cases.IMPORTANCEThe JI Panel demonstrates high sensitivity for acute PJI but lower sensitivity for chronic infections. The ability of the JI Panel to rapidly identify pathogens in acute cases plays a significant role in improving antibiotic stewardship, ensuring timely and appropriate treatment. Given the lower sensitivity for chronic PJI, further research could focus on improving the detection of pathogens that are commonly involved in chronic infections. While the JI Panel is a promising tool for acute PJI diagnosis and supports rapid antibiotic stewardship, its limitations in chronic cases and under antibiotic exposure must be addressed to maximize its clinical utility.

The widespread adoption of metagenomic next-generation sequencing has revolutionized microbial detection, yet contaminating DNA in laboratory reagents poses significant challenges for result interpretation. This study investigated microbial contamination profiles across four commercial DNA extraction reagent brands (M, Q, R, and Z) and assessed batch-to-batch variability. Extraction blanks were generated using molecular-grade water or ZymoBIOMICS Spike-in Control I as input materials. Analysis revealed distinct background microbiota profiles between brands, with some containing common pathogenic species that could affect clinical interpretation. Notably, background contamination patterns varied significantly between different lots of the same brand, highlighting the need for lot-specific microbiota profiling. Site-specific environmental contaminants were identified through analysis of 30 control samples from a single study site. Additionally, comparison of blood samples from healthy individuals with control samples suggested no evidence of a consistent blood microbiome, suggesting that "extraction blanks" may serve as negative controls in clinical metagenomic testing of sterile liquid biopsy samples. These findings emphasize the importance of including negative controls in every run and underscore the need for manufacturers to provide comprehensive background microbiota data for each reagent lot to optimize clinical interpretation and minimize false-positive results. IMPORTANCE:Metagenomic next-generation sequencing (mNGS) has revolutionized pathogen detection and microbiome studies, but contamination from DNA extraction reagents remains a critical challenge. This study highlights the significant variability in background microbiota profiles across reagent brands and manufacturing lots, emphasizing the need for manufacturers to provide detailed contamination profiles. Our findings underscore the importance of implementing extraction blanks as standard controls and incorporating bioinformatics tools to account for background noise. These measures are essential to enhance the reliability of mNGS results and prevent diagnostic errors, particularly in clinical settings where contamination could mask or mimic pathogen signals. Additionally, our confirmation that healthy blood lacks a consistent microbiome helps streamline control selection in clinical testing protocols, potentially reducing costs and complexity in clinical mNGS workflows.