BACKGROUND:Multiplex nucleic acid amplification tests (NAATs) are essential for managing respiratory viral infections. To robustly confirm the capability of laboratories for multiplex respiratory viral detection, a well-designed, large-scale external quality assessment study incorporating in-depth analysis was conducted. METHODS:Laboratories performing multiplex viral NAATs in China were publicly recruited and provided with a reference panel to assess their testing performance. Multivariate analysis was performed on the collected results and detailed methodological data to identify potential sources of errors. RESULTS:Among 914 participating laboratories, overall concordance was 81.6% (746/914). False-positives (FPs) were concentrated in 13 laboratories, suggesting the occurrence of laboratory contamination. False-negatives (FNs) were more prevalent, primarily occurring in samples with low viral loads. Multivariate analysis identified capillary electrophoresis-based multiplex PCR assay (aOR = 2.1, 95% CI: 1.1-3.8), certain commercial kit (Flu/RSV kit [Autobio], aOR = 102.2, 95% CI: 13.4-776.0), and laboratory-developed tests (aOR = 6.59, 95% CI: 2.2-19.8) as independent risk factors for FNs. Suboptimal laboratory performance also increased the odds of FNs. CONCLUSIONS:This study demonstrates the advances and limitations of multiplex viral NAATs: high adoption and acceptable concordance reflect the technology's maturity, yet the occurrence of FNs and FPs highlights a need for continued assay optimization, strengthened laboratory quality management, and increased clinician awareness of testing limitations.

Background:Coronary venous ethanol ablation (CVEA) is increasingly used for the treatment of ventricular arrhythmias (VAs). Objective:This study aimed to characterize the features of VAs eliminated by CVEA. Methods:Consecutive patients who underwent CVEA were prospectively enrolled and classified as acute success or failure groups. Targeted veins were categorized as the first septal vein (S1), left ventricular annular vein (LVA), or anterior interventricular vein (AIV). Results:42 patients (mean age 52.5 ± 14.3 years; 42.9% female) underwent CVEA; 29 (69.0%) achieved acute success. Ethanol was delivered via S1 in 16 patients, LVA in 8, and AIV in 5. In success cases, S1- and LVA-eliminated VAs exhibited similar right bundle branch block-like morphologies, whereas AIV-related VAs showed a left bundle branch block pattern with QS in leads V1-V2 and late precordial R-wave transition. During ethanol infusion, a transient "irritative" response in VA burden occurred in 44.8% of patients and was more frequently observed in successful cases. Complications were limited to transient bundle branch block in 4 of 42 (9.5%) and transient ST-T changes in 1 of 42 (2.4%), which resolved spontaneously after pausing/terminating ethanol infusion as needed. During follow-up, 4 patients experienced VA recurrences. One underwent repeat CVEA at the same target vein, and 3 were successfully managed with drug therapy. Conclusion:Successful CVEA from S1 and LVA yield similar electrocardiographic morphologies, whereas AIV-related VAs were characterized by QS in V1-V2 with late precordial transition. An irritative response during ethanol infusion may indicate effective ablation, and ethanol delivery from S1 or LVA may induce transient bundle branch block.

Acute coronary syndrome triage at first medical contact begins with patient narratives, while definitive diagnosis often requires waiting for cardiac troponin or electrocardiogram results. We developed TriageMaster-70B, a large language model using only patient narratives and vital signs for the triage. In 16,428 retrospective and 512 prospective cases, it showed high sensitivity, with per-case processing 39% faster than independent retrospective cardiologist review. These findings suggest TriageMaster-70B may support rapid, accurate, and interpretable triage in emergency departments. Clinical trial registration: ClinicalTrials.gov NCT06493175; first posted September 20, 2024.

BACKGROUND:It is assumed that complete balloon occlusion is necessary for effective lesion formation in retrograde coronary venous ethanol infusion for ventricular arrhythmias. OBJECTIVE:This study aimed to evaluate the myocardial effects of ethanol infusion under conditions of incomplete balloon occlusion. METHODS:12 swine were randomized into acute (n = 6) and chronic (n = 6) cohorts. Ethanol was infused into different coronary veins using single over-the-wire balloons sized to achieve incomplete occlusion. Myocardial injury was assessed by gross pathology, histology, and ultrastructural analysis. RESULTS:Incomplete balloon occlusion was confirmed in all animals, with distal venous leakage in 9 of 12 and collateral filling in 3 of 12. Myocardial staining was observed in only 4 of 12, faint and poorly localized. Despite this, all acute animals developed lesions, with transmural infarction in 5 of 6. Gross lesion dimensions reached up to 30 mm in width, 86 mm in length, and 15.0 mm in depth. Acute histology revealed eosinophilic changes, vacuolar degeneration, and cardiomyocyte necrosis. Ultrastructural analysis demonstrated plasma membrane rupture, sarcomeric dissolution, mitochondrial swelling, and intercalated disk disruption. In the chronic phase, 4 of 6 swine presented with transmural fibrotic scars (up to 46 mm wide, 58 mm long, and 14.8 mm deep), characterized by replacement fibrosis, myocyte atrophy, and inflammation. Chronic ultrastructural changes included mitochondrial degeneration, intercalated disk remodeling, sarcomere disorganization, and vacuolar pathology. CONCLUSION:Incomplete balloon occlusion during retrograde coronary venous ethanol infusion can still produce large, transmural infarctions. These findings underscore the potential for off-target necrosis and the need for precise procedural control in ethanol ablation for ventricular arrhythmias.

BACKGROUND:Nanosecond pulse field ablation (nanoPFA) is an emerging non-thermal modality that may reduce muscle contractions and enable atrial fibrillation (AF) ablation under conscious sedation. OBJECTIVE:This study aimed to compare the muscle response, safety, and 6-month outcomes between nanoPFA and microsecond PFA (microPFA). METHODS:Patients with symptomatic paroxysmal AF underwent nanoPFA under conscious sedation or microPFA under general anesthesia. In the nanoPFA group, muscle contraction, pain (Visual Analogue Scale [VAS]), and patient experience (Likert scale questionnaire) were assessed. Outcomes were evaluated at 3 and 6 months. Propensity score matching (PSM) was performed as sensitivity analysis. RESULTS:Among 151 patients (60.2 ± 11.4 years, 32.5% female), 57 received nanoPFA and 94 underwent microPFA. Pulmonary vein and superior vena cava isolation was completed in all, with no device-related complications. During nanoPFA, muscle contraction was absent in 43.9%, slight in 47.4%, and severe in 8.8%, most frequently at the right superior pulmonary vein. Most patients reported mild/no discomfort (VAS 0-4: 71.9% [41/57]); none reported severe pain, and 87.7% preferred conscious sedation. Six-month Kaplan-Meier freedom from any atrial tachyarrhythmia was 91.8% with nanoPFA and 83.9% with microPFA (log-rank P = .57); after PSM, 6-month freedom remained similar between groups (90.0% vs 88.9%, log-rank P = .56). CONCLUSION:NanoPFA performed under conscious sedation provides comparable safety and similar 6-month arrhythmia outcomes to microPFA under general anesthesia, with minimal pain and muscle responses, supporting the feasibility of a conscious-sedation workflow in our center.

BACKGROUND:Microsecond pulsed field ablation (PFA) (microPFA) has been associated with postablation hemolysis, whereas evidence for the use of nanosecond PFA (nanoPFA) remains limited. OBJECTIVE:This study aimed to compare hemolysis and acute kidney injury (AKI) after microPFA, nanoPFA, and radiofrequency ablation (RFA). METHODS:We included 323 patients who underwent atrial fibrillation ablation at a high-volume Chinese center, including microPFA (n = 186), nanoPFA (n = 65), or RFA (n = 72). Pulmonary vein isolation was performed with or without additional ablation. Hemolysis and renal biomarkers were measured at baseline, procedure end, and 24 hours after the procedure. RESULTS:PFA produced obvious hemolysis, which was not observed with RFA. Postablation bilirubin, lactate dehydrogenase (LDH), and free hemoglobin increased and haptoglobin decreased with PFA. LDH (229.0 ± 50.6 vs 198.4 ± 33.8 IU/L; P < .001) and free hemoglobin levels (289.3 ± 45.1 vs 263.7 ± 55.0 ng/mL; P = .043) were higher with microPFA than with nanoPFA after multivariable adjustment. Hemoglobin decline and hemoglobin drop of >20 g/L occurred more often after microPFA (44.1% and 58.1%) than after RFA (22.2% and 27.8%) and nanoPFA (18.5% and 20.0%). AKI was uncommon (microPFA 3; nanoPFA 1; RFA 0). In microPFA, hemolysis scaled with delivery number (LDH ratio r = 0.26; 95% confidence interval 0.10-0.40; bilirubin ratio r = 0.15; 95% confidence interval 0-0.29), whereas no dose-dependent association was detected with nanoPFA. CONCLUSION:PFA for atrial fibrillation typically induced hemolysis; however, the application-related dose effect was observed in microPFA, but not in nanoPFA. NanoPFA was associated with lower hemolysis indices, whereas severe anemia and AKI may be rare. Larger studies are needed to confirm modality differences, define clinical impact, and optimize application titration.

Objective Heart Failure with preserved Ejection Fraction(HFpEF)is highly prevalent among patients with Hypertrophic CardioMyopathy(HCM),and early identification is critical for improving disease management.However,early screening for HFpEF remains challenging because symptoms are non-specific,diagnostic procedures are complex,and follow-up costs are high.Smartphones,owing to their wide accessibility,low cost,and portability,provide a feasible means to support heart sound-based screening.In this study,smartphone-acquired heart sounds from patients with HCM are used to develop and train an ensemble learning classification model for early detection and dynamic self-monitoring of HFpEF in the HCM population. Methods The proposed HFpEF screening framework consists of three components:preprocessing,feature extraction,and model training and fusion based on ensemble learning(Fig.1).During preprocessing,smartphone-acquired heart sounds are subjected to bandpass filtering and wavelet denoising to improve signal quality,followed by segmentation into individual cardiac cycles.For feature extraction,Mel-Frequency Cepstral Coefficients(MFCCs)and Short-Time Fourier Transform(STFT)time-frequency spectra are calculated(Fig.3).For classification,a stacking ensemble strategy is applied.Base learners,including a Support Vector Machine(SVM)and a Convolutional Neural Network(CNN),are trained,and their predicted probabilities are combined to construct a new feature space.A Logistic Regression(LR)meta-learner is then trained on this feature space to identify HFpEF in patients with HCM. Results and Discussions The classification performance of the three models is evaluated using the same patient-level independent test set.The SVM base learner achieves an Area Under the Curve(AUC)of 0.800,with an accuracy of 0.766,sensitivity of 0.659,and specificity of 0.865(Table 5).The CNN base learner attains an AUC of 0.850,with an accuracy of 0.789,sensitivity of 0.622,and specificity of 0.944(Table 5).By comparison,the ensemble-based LR classifier demonstrates superior performance,reaching an AUC of 0.900,with an accuracy of 0.813,sensitivity of 0.768,and specificity of 0.854(Table 5).Relative to the base learners,the ensemble model exhibits a significant overall performance improvement after probability-based feature fusion(Fig.5).Compared with existing clinical HFpEF risk scores,the proposed method shows higher predictive performance and stronger dynamic monitoring capability,supporting its suitability for risk stratification and follow-up warning in home settings.Compared with professional heart sound acquisition devices,the smartphone-acquired approach provides greater accessibility and cost efficiency,supporting its application in auxiliary HFpEF screening for high-risk HCM populations. Conclusions The challenges of clinical HFpEF screening in patients with HCM are addressed by proposing a smartphone-acquired heart sound analysis approach combined with an ensemble learning prediction model,resulting in an accessible and easily implemented auxiliary screening pipeline.The effectiveness of smartphone-based heart sound analysis for initial HFpEF screening in patients with HCM is validated,demonstrating its feasibility as an economical auxiliary tool for early HFpEF detection.This approach provides a non-invasive,convenient,and efficient screening strategy for patients with HCM complicated by HFpEF.

Large language models (LLMs) are rapidly advancing medical artificial intelligence, offering revolutionary changes in health care. These models excel in natural language processing (NLP), enhancing clinical support, diagnosis, treatment, and medical research. Breakthroughs, like GPT-4 and BERT (Bidirectional Encoder Representations from Transformer), demonstrate LLMs’ evolution through improved computing power and data. However, their high hardware requirements are being addressed through technological advancements. LLMs are unique in processing multimodal data, thereby improving emergency, elder care, and digital medical procedures. Challenges include ensuring their empirical reliability, addressing ethical and societal implications, especially data privacy, and mitigating biases while maintaining privacy and accountability. The paper emphasizes the need for human-centric, bias-free LLMs for personalized medicine and advocates for equitable development and access. LLMs hold promise for transformative impacts in health care.

Telomere-to-telomere (T2T) genome assemblies are indispensable for accurate detection of genetic variation and for resolving complex repetitive regions. Monozygotic (MZ) twin pedigrees provide a powerful model to investigate de novo mutations (DNMs), however, comprehensive, haplotype-resolved analyses of structural variation (SV), allele-specific inheritance in complex regions, and DNA methylation in diploid human genomes remain limited. Here, we generated complete, haplotype-resolved T2T assemblies for two female twins (C33 and C35) from a Han Chinese pedigree by integrating complementary, state-of-the-art sequencing technologies. The resulting T2T-C33 and T2T-C35 assemblies are highly contiguous and complete, with Genome Continuity Inspector (GCI) scores of 74.94 (maternal) and 77.94 (paternal), and consensus quality values (QV) >75 ( k = 21). We comprehensively cataloged 62 inter-twin single-nucleotide variants (SNVs), 15 small indels, and identified both shared and private DNMs, revealing nascent genomic divergence between the MZ twins. Focused interrogation of complex regions uncovered pronounced haplotype-specific length polymorphisms and structural heterogeneity within centromeric higher-order repeat (HOR) arrays. Notably, we observed extensive HOR copy-number variation between haplotypes, including a large copy-number difference on maternal chromosome 18, underscoring dynamic HOR array evolution even among genetically identical individuals. Concurrently, genome-wide DNA methylation profiling delineated allele-specific epigenetic variation that may contribute to phenotypic discordance. Together, these high-quality, diploid T2T genomes from a Han Chinese pedigree provide a valuable resource for population-aware genomics and reveal fine-scale, haplotype-specific divergence in MZ twins. Our results advance understanding of repeat dynamics, centromeric architecture, epigenetic variation and the spectrum of human genomic variation at single-base and structural scales. ### Competing Interest Statement The authors have declared no competing interest. National Key Research and Development Program of China, 2024YFC3405701, 2025YFC3410300, 2022YFC3400304 National High Level Hospital Clinical Research Funding, BJ-2025-177

The ability to detect specific DNA, including single nucleotide variants (SNVs), with high sensitivity is essential for advancing genetic research, diagnostics, and personalized medicine. This study presents a novel method for ultrasensitive DNA detection, combining ligation-rolling circle amplification (L-RCA) with CRISPR-Cas12a. While L-RCA systems have been widely used for nucleic acid detection, the sensitivity of conventional L-RCA generally reaches approximately 100 pM. Here, we demonstrate that the sensitivity of RCA-Cas12a systems can be markedly enhanced by incorporating multiple CRISPR target regions into the padlock probe. This method achieves remarkable sensitivity, detecting DNA at concentrations as low as 1 aM (6 copies per reaction), and is capable of identifying single nucleotide variants (SNVs) with allele fractions as low as 1 %. Unlike many current complex RCA-Cas12a strategies, this approach is simple and does not require advanced labeling or instrumentation, making it a promising tool for ultrasensitive DNA detection in various applications.

BACKGROUND:Circulating tumor DNA (ctDNA) testing of plasma for ESR1 somatic variants is essential for guiding treatment decisions in hormone receptor-positive (HR  +  ) and HER2-negative (HER2-) advanced or metastatic breast cancer (MBC) patients who have progressed on frontline therapy. To ensure optimal, uniform, and reliable ESR1 testing across China, an pilot external quality assessment (EQA) scheme was established. METHODS:Aliquots of five artificial reference plasma samples containing ESR1 mutations at varying allelic frequencies were distributed to 37 laboratories for testing and reporting according to routine procedures. The genotyping accuracy and clinical reporting were evaluated against standardized criteria, and feedback was provided to the participants. RESULTS:The overall genotyping error rate in the EQA was 6.29%, with 91.4% of laboratories correctly identifying the ESR1 mutational status in all samples. A variety of extraction methods and analytical techniques were employed. However, reports often failed to address the risk that tumor DNA may not have been tested, and the limitations of the methodologies used by participants were insufficiently discussed. CONCLUSION:The variability in genotyping accuracy and reporting standards underscores the importance of EQA and educational guidance to ensure the provision of high-quality clinical services.

[This corrects the article DOI: 10.1371/journal.pone.0171991.].

Background:It remains uncertain whether a novel composite indicator, the high sensitivity C-reactive protein (CRP) (hsCRP)-to-body mass index (BMI) ratio (CBR), is associated with unfavourable events in subjects who have undergone drug-eluting stent (DES) implantation. Therefore, we conducted a prospective cohort study to evaluate the associations between the CBR and adverse outcome occurrences. Methods:From January 2013 to December 2013, a total of 9810 subjects who underwent DES implantation were enrolled in the study. The participants were divided into three groups on the basis of the CBR. We defined the primary endpoint as the occurrence of major adverse cardiovascular and cerebrovascular events (MACCEs), and the secondary endpoints included the occurrence of all-cause death, myocardial infarction(MI), stroke or target vessel revascularization (TVR). Kaplan‒Meier (K‒M) analysis and Cox regression analysis were performed to assess the discrepancy in the risk of adverse outcomes between the different CBR groups. Results:Over an average follow-up period of 26.7 months, 1022 MACCEs were recorded, which included 122 deaths, 392 MIs, 156 strokes and 461 instances of TVR. The results from the K‒M analysis suggested that the rates of MACCEs and MI increased with increasing tertiles of the CBR. Furthermore, Cox regression analysis demonstrated that there were significant associations between a high CBR and increased MACCE and MI risk. Conclusion:In subjects who underwent DES implantation, a higher CBR was significantly related to increased long-term MACCE and MI risk.

Venous ethanol ablation (VEA) is effective for ablation-refractory ventricular arrhythmias (VAs), particularly those arising from the left ventricular (LV) summit and interventricular septum 1 .However, numerous intricate veins within this region make the accurate mapping technique-challenging 2 .Despite the microelectrode or guidewire could

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

Background: Provisional T-stenting is generally acknowledged for treating coronary bifurcation lesions, but risk of adverse cardiac events is still high. Drug-coated balloon (DCB) has been proved non-inferior and even superior to stent combined with general balloon strategy but the efficacy and safety of using it alone in bifurcation lesions needs further investigation. Hence, this prospective, multi-center, randomized, controlled study was devised to test whether DCB-only technique is non-inferior to provisional T-stenting with drug-eluting stent (DES) for true coronary bifurcation lesions. Methods: A total of 1000 patients with true coronary bifurcation lesions (Medina classes 1.1.1, 1.0.1 or 0.1.1) from 20 research institutes in China who meet the inclusion criteria and satisfy no exclusion criteria will be enrolled in this trial. Subjects will be randomly assigned to the RESTORER DCB group or the DES group in a 1:1 ratio. Patients in the DCB group will receive DCB treatment only, while those in the DES group will undergo provisional T-stenting. Coronary angiography will be performed by well-trained operators and participants will take antiplatelet or anticoagulation medication in accordance with the protocol. The first primary endpoint is the target lesion failure (TLF) rate at 12 months postoperatively. The second primary endpoint is clinical net event rates at 12 months postoperatively. The follow-up data will be collected at 1, 6 and 12months and 2 and 3 years postoperatively by phone call or clinic visit. Statistical analysis will test the noninferiority of DCB regarding the first primary endpoint and further test the superiority of DCB concerning the second primary endpoint if the noninferiority is supported. Discussion: This trial is intended to test whether DCB-only technique is non-inferior to provisional T-stenting with DES in true coronary bifurcation lesions, which will provide evidence for strategy selection in treating such patients. Trial registration: NCT04842838 [ClinicalTrials.gov] [registered on April 13, 2021]

COVID-19, caused by SARS-CoV-2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR-Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in-depth examination of the applications of CRISPR-Cas in mutation detection specifically for SARS-CoV-2. It begins by introducing SARS-CoV-2 and existing variant detection platforms. The principles of the CRISPR-Cas system are then clarified, followed by an exploration of three CRISPR-Cas-based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR-Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR-Cas system is provided.

Objective: This study aimed to identify key prognostic factors in patients with hypertrophic cardiomyopathy (HCM) presenting heart failure with preserved ejection fraction (HFpEF), with a special focus on the role of Free Triiodothyronine (FT3) levels. Methods: A retrospective cohort study was conducted, comprising HFpEF patients admitted to two medical centers from 2009 to 2019. Data collection included demographic and clinical parameters, particularly FT3 levels. Univariate and multivariate Cox analyses, Kaplan-Meier (KM) survival curve analysis, and Restricted Cubic Spline (RCS) curve analysis were employed to identify prognostic factors and to assess the non-linear predictive value of FT3. Results: The analysis revealed that age, atrial fibrillation, and NT-proBNP levels significantly influenced prognosis in heart failure patients. Additionally, FT3 levels emerged as a significant independent predictor of both all-cause mortality and cardiac transplantation. Patients with lower FT3 levels exhibited poorer long-term prognoses, with 2.885 identified as the critical FT3 value. Conclusions: These findings underscore the importance of FT3 levels as a crucial prognostic factor in patients with heart failure, warranting consideration in patient outcomes assessments. Clinicians are recommended to monitor FT3 levels and contemplate interventions aimed at maintaining or enhancing thyroid function in these patients to improve outcomes.