SEARCH · Search PubMed

Results for “culture-negative”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

8 recordsLinked to original sources

PubMed2026

Computed tomography-guided precision biopsy combined with metagenomic next-generation sequencing for etiological diagnosis in patients with blood culture-negative systemic infections.

ObjectiveTo evaluate the diagnostic efficacy of computed tomography-guided percutaneous biopsy combined with metagenomic next-generation sequencing in patients with blood culture-negative systemic infections and to assess the clinical impact of using this combined strategy for etiological confirmation and guidance of targeted antimicrobial therapy.MethodsThis single-center retrospective observational cohort study enrolled 78 patients who met the Sepsis-3 consensus criteria for suspected systemic infection and had negative conventional microbiological work-ups (at least two sets of blood cultures) between April 2022 and March 2025. All patients underwent computed tomography-guided biopsy of radiologically identified infectious foci, with specimens processed concurrently for conventional culture and metagenomic next-generation sequencing. Diagnostic performance was benchmarked against the final comprehensive clinical diagnosis, and the influence of metagenomic next-generation sequencing findings on antimicrobial therapy modification was analyzed. Sample size calculation, based on a prior study estimating an metagenomic next-generation sequencing detection rate of 85% (α = 0.05, β = 0.2), indicated a minimum of 68 cases; accordingly, 78 patients were enrolled.ResultsComputed tomography-guided biopsy was technically successful in all 78 patients (100%). The pathogen detection rate of metagenomic next-generation sequencing (91.0%, 71/78) was significantly higher than that of conventional culture (55.1%, 43/78; p < 0.001). Using the final clinical diagnosis as the reference standard, metagenomic next-generation sequencing achieved a sensitivity of 94.7% (95% confidence interval: 86.9-98.5), specificity of 100.0% (95% confidence interval: 29.2-100.0), positive predictive value of 100.0% (95% confidence interval: 94.9-100.0), and negative predictive value of 42.9% (95% confidence interval: 9.9-81.6). Among the 35 culture-negative specimens, metagenomic next-generation sequencing established a definitive microbiological diagnosis in 28 cases (80.0%) and detected polymicrobial infections in 11 cases (14.1% of the cohort). Antimicrobial therapy was rationally adjusted based on metagenomic next-generation sequencing results in 69.2% (54/78) of the patients.ConclusionsThe integration of computed tomography-guided precision biopsy with metagenomic next-generation sequencing offers a highly effective diagnostic approach for blood culture-negative systemic infections. This synergistic strategy improves etiological diagnosis by providing high-yield target specimens that enable comprehensive, unbiased pathogen screening, facilitates differentiation between infectious and non-infectious etiologies, and supplies critical evidence for guiding precision antimicrobial therapy. These findings highlight the growing role of interventional radiology in the contemporary framework of precision infectious disease management.

Humans↗

PubMed2026

The Next Step: The Role of Metagenomic Next-Generation Sequencing in Microbial Detection of Culture-Negative Cardiovascular Infections.

Cardiovascular infections, including those that involve native and prosthetic heart valves, implantable cardiac devices, mechanical circulatory assist devices, and vascular grafts, are associated with significant morbidity and mortality risks. Optimal management of these complex infections requires pathogen-directed antimicrobial therapy. However, standard culture-based methods often fail to identify causative organisms due to prior antimicrobial use, infections due to fastidious organisms, or biofilm-associated infections. Emerging evidence suggests that microbial cell-free DNA (mcfDNA) and metagenomic testing can enhance pathogen detection, particularly in culture-negative cases. However, their results require careful clinical interpretation, often necessitating input from infectious diseases specialists. In this review, we examine published evidence regarding metagenomic testing for cardiovascular infections and its impact on patient care. We propose a framework for microbiological adjudication of mcfDNA results, introduce standardized definitions for clinical impact assessment, and provide guidance on integrating mcfDNA testing into diagnostic evaluation of patients with culture-negative cardiovascular infections.

Humans↗

PubMed2026

Diagnostic Accuracy of a CRISPR-Based Assay in Smear- and Culture-Negative Fungal Keratitis.

IMPORTANCE: Diagnosing fungal keratitis (FK) in patients with negative smear and culture results remains clinically challenging, highlighting the need for alternative diagnostic approaches. OBJECTIVE: To determine the diagnostic accuracy of the clustered regularly interspaced short palindromic repeats (CRISPR)-based Rapid Identification of Mycoses using CRISPR (RID-MyC) assay for detecting FK in patients with negative smear and culture results using in vivo confocal microscopy (IVCM) as the reference standard. DESIGN, SETTING, AND PARTICIPANTS: This prospective diagnostic accuracy study was conducted from December 2024 to March 2025 at Aravind Eye Hospital, a tertiary ophthalmology referral hospital in Coimbatore, India. Consecutive patients clinically suspected to have microbial keratitis with negative smear and culture results were eligible for inclusion. Data were analyzed from March 2025 to June 2025. INTERVENTIONS: All included participants underwent corneal scraping for RID-MyC assay and imaging by IVCM. MAIN OUTCOMES AND MEASURES: The primary outcomes were sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic concordance of the RID-MyC assay compared with IVCM results. RESULTS: Of 245 consecutive patients clinically suspected to have microbial keratitis, 82 were smear negative. After exclusions due to contraindications or positive subsequent cultures, 41 patients with smear- and culture-negative results were ultimately included in the final analysis. Of these 41 patients (mean [SD] age, 51.0 [14.6] years; 21 [51.2%] women), RID-MyC demonstrated sensitivity of 82.1% (95% CI, 63%-94%) and specificity of 76.9% (95% CI, 46%-95%). Positive predictive value was 88.5% (95% CI, 74%-95%) and negative predictive value was 66.7% (95% CI, 46%-82%). Concordance between RID-MyC and IVCM was observed in 33 cases (80.5%). Notably, prior antifungal treatment was most frequent (4 of 5 [80%]) among patients with positive IVCM but negative RID-MyC results. Conversely, all patients (3 of 3 [100%]) with negative IVCM but positive RID-MyC findings had smaller, peripheral, or paracentral lesions. CONCLUSIONS AND RELEVANCE: In this diagnostic study, in patients with smear- and culture-negative FK, the RID-MyC assay showed good diagnostic accuracy comparable with IVCM and was feasible in all cases, including those in whom imaging was not possible. With its rapid turnaround and minimal equipment needs, RID-MyC may serve as a practical adjunct to conventional diagnostics, particularly in high-burden, resource-limited settings where IVCM is unavailable or contraindicated.

Humans↗

PubMed2026

The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection.

Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10⁵ CFU/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as Escherichia coli. When evaluated against conventional cultures using 95 sonicate fluid and 276 pre/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level Staphylococcus. Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying Staphylococcus previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.IMPORTANCENext-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.

Humans↗

PubMed2026

Rapid diagnosis of common, undetected, and uncultivable bloodstream infections from positive blood cultures using Oxford Nanopore sequencing: a metagenomic pipeline analysis.

BACKGROUND: Metagenomic sequencing can potentially transform clinical microbiology by enabling rapid pathogen identification and antimicrobial resistance (AMR) prediction in critically ill patients with bloodstream infections. However, the clinical use of metagenomic sequencing has been constrained by its speed, accuracy, and technical feasibility. Our aim was to develop and evaluate a direct-from-positive blood culture workflow using Oxford Nanopore sequencing that overcomes these limitations and delivers rapid, accurate results. METHODS: In this metagenomic pipeline analysis, 211 positive (130 aerobic and 81 anaerobic) and 62 negative (30 aerobic and 32 anaerobic) randomly selected blood cultures were processed from Oxford University Hospitals for comparing species identification, AMR detection, and time-to-result against standard culture-based diagnostics performed by the hospital's routine microbiology laboratory. Species prediction was performed using Kraken2 with a comprehensive standard database, applying heuristic and random forest classification models. Additionally, we benchmarked AMR classification tools and databases, including ResFinder, CARD, and NCBI AMRFinderPlus. FINDINGS: Across all samples, our method achieved 97% sensitivity and 94% specificity for species identification compared with that of routine culture and matrix-assisted laser desorption ionisation time-of-flight-based diagnostics; both sensitivity and specificity increased to 100% after adjudication of plausible additional infections. We detected 19 additional infections (13 polymicrobial, five previously unidentifiable, and one in a culture-negative sample) and delivered species identification results within 3 h 20 min (IQR 3 h 7 min-3 h 27 min), approximately 10 h earlier than routine diagnostic methods. For the ten most common clinically relevant pathogens, our method yielded AMR results 20 h earlier than current antimicrobial susceptibility testing, with an overall sensitivity of 88% and specificity of 93%. Performance varied by species. For Staphylococcus aureus, the AMR prediction sensitivity was 100% and specificity was 99%, and for Escherichia coli, the prediction sensitivity was 91% and specificity was 94%. INTERPRETATION: These findings show that metagenomic sequencing has the potential to rapidly and comprehensively detect pathogens and AMR in bloodstream infections. Integration into clinical practice could help to close diagnostic gaps, reduce empirical antibiotic use, and enable rapid targeted treatment. Nonetheless, improvements in AMR prediction for some species and drugs, along with further multisite validation, are required before clinical implementation. FUNDING: National Institute for Health Research (NIHR) Oxford Biomedical Research Centre.

Humans↗

PubMed2025

Real-world clinical impact of plasma cell-free DNA metagenomic next-generation sequencing assay.

OBJECTIVE: To describe the real-world clinical impact of a commercially available plasma cell-free DNA metagenomic next-generation sequencing assay, the Karius test (KT). METHODS: We retrospectively evaluated the clinical impact of KT by clinical panel adjudication. Descriptive statistics were used to study associations of diagnostic indications, host characteristics, and KT-generated microbiologic patterns with the clinical impact of KT. Multivariable logistic regression modeling was used to further characterize predictors of higher positive clinical impact. RESULTS: We evaluated 1000 unique clinical cases of KT from 941 patients between January 1, 2017-August 31, 2023. The cohort included adult (70%) and pediatric (30%) patients. The overall clinical impact of KT was positive in 16%, negative in 2%, and no clinical impact in 82% of the cases. Among adult patients, multivariable logistic regression modeling showed that culture-negative endocarditis (OR 2.3; 95% CI, 1.11-4.53; P .022) and concern for fastidious/zoonotic/vector-borne pathogens (OR 2.1; 95% CI, 1.11-3.76; P .019) were associated with positive clinical impact of KT. Host immunocompromised status was not reliably associated with a positive clinical impact of KT (OR 1.03; 95% CI, 0.83-1.29; P .7806). No significant predictors of KT clinical impact were found in pediatric patients. Microbiologic result pattern was also a significant predictor of impact. CONCLUSIONS: Our study highlights that despite the positive clinical impact of KT in select situations, most testing results had no clinical impact. We also confirm diagnostic indications where KT may have the highest yield, thereby generating tools for diagnostic stewardship.

Humans↗

PubMed2026

Integrating metagenomic next-generation sequencing into a multimodal diagnostic framework for spinal infection: enhancing etiological identification and clinical prediction.

BACKGROUND: Spinal infection (SI) remains diagnostically challenging because of heterogeneous etiologies, nonspecific clinical manifestations, and the limited sensitivity of conventional microbiological approaches, particularly following empirical antimicrobial exposure. Although metagenomic next-generation sequencing (mNGS) enables unbiased pathogen detection, its incremental clinical value beyond pathogen identification and its role within integrated diagnostic strategies remain incompletely established. METHODS: We retrospectively analyzed 208 consecutive patients with suspected SI between August 2022 and August 2025. Final diagnoses were established using a multidisciplinary-adjudicated composite reference standard incorporating clinical, radiological, microbiological, and histopathological evidence. The diagnostic performance of mNGS was compared with conventional culture and histopathology. Furthermore, multimodal predictive models integrating clinical variables and microbiological information were developed using L1-regularized logistic regression. RESULTS: In the comparative cohort, mNGS achieved a significantly higher diagnostic yield than culture (66.5% vs. 27.41%, P < 0.001). Among confirmed SI cases, mNGS demonstrated higher sensitivity than conventional culture (91.67% vs. 40.15%, P < 0.001). mNGS identified a substantially broader pathogen spectrum, ranging from fastidious organisms such as Mycobacterium tuberculosis and Brucella to rare pathogens including Talaromyces marneffei and Coxiella burnetii, and maintained robust sensitivity (98.2%) despite prior antibiotic exposure. While an integrated clinical model achieved an AUC of 0.916, mNGS as a standalone modality provided superior discriminative power (AUC = 0.889) compared to histopathology (AUC = 0.836), the Conventional Biomarker Model (AUC = 0.742), and culture (AUC = 0.693). CONCLUSIONS: mNGS is a high-yield diagnostic tool for spinal infection, particularly in culture-negative and antibiotic-pretreated scenarios. Integrating mNGS into a multimodal clinical framework facilitates etiological clarity and precision antimicrobial therapy.

Humans↗

PubMed2026

Lawsonella clevelandensis: a normal flora that bites deep.

Since its formal description in 2016, Lawsonella clevelandensis-a strictly anaerobic, partially acid-fast bacterium-has been increasingly recognized as a cause of deep-seated abscesses, yet its fastidious nature and absence from routine diagnostic databases contribute to significant underdiagnosis. This narrative review synthesizes current knowledge on its microbiology, expanding clinical spectrum, diagnostic strategies, and treatment, based on a literature search of PubMed and Web of Science up to April 2026. Analysis of 27 documented publications, comprising 18 clinical cases, reveals a potential association with host risk factors including diabetes, immunosuppression, and prior surgical procedures, alongside a notable predilection for fat-rich tissues such as the breast and abdomen. While metagenomic next-generation sequencing and 16S rRNA gene amplification have become indispensable for definitive identification, antimicrobial susceptibility data-derived primarily from a single strain-demonstrate uniformly low minimum inhibitory concentrations for penicillins, carbapenems, clindamycin, and metronidazole, with no acquired resistance genes identified by whole-genome sequencing. However, the absence of established clinical breakpoints and limited tested isolates precludes definitive conclusions about universal susceptibility. Clinicians should maintain a high index of suspicion for L. clevelandensis in culture-negative deep abscesses, particularly those with acid-fast rods, as prompt diagnosis and empirical therapy with β-lactam/β-lactamase inhibitors or carbapenems appear reasonable based on current in vitro and clinical evidence, though further susceptibility surveillance is essential.

Humans↗