Search Results (2,697)

Tumor mutational burden (TMB) is a key pan-cancer biomarker for immunotherapy selection, but its routine assessment by whole-exome sequencing (WES) or large next-generation sequencing (NGS) panels is costly, time-consuming, and constrained by tissue and DNA quality. In parallel, advances in computational pathology have enabled deep learning models to infer molecular biomarkers directly from hematoxylin and eosin (H&E) whole-slide images (WSIs), raising the prospect of a purely digital assay for TMB. In this comprehensive review, we surveyed PubMed and Scopus (2015–2025) to identify original studies that applied deep learning directly to H&E WSIs of human solid tumors for TMB estimation. Across the 17 eligible studies, deep learning models have been applied to predict TMB from H&E WSIs in a variety of tumors, achieving moderate to good discrimination for TMB-high versus TMB-low status. Multimodal architectures tended to outperform conventional CNN-based pipelines. However, heterogeneity in TMB cut-offs, small and imbalanced cohorts, limited external validation, and the black-box nature of these models limit clinical translation. Full article

The use of antiretroviral therapy (ART) as the only effective way to control human immunodeficiency virus (HIV) infection results in HIV drug resistance. Next-generation sequencing (NGS) has become a common method for identifying drug-resistant variants and reducing analysis costs. The aim of this study was to develop an NGS-based protocol for identifying resistance mutations and cell tropism of HIV-1 in adult patients with and without treatment experience in Russia in 2024–2025. Plasma samples from adult HIV-infected patients from Russia were analyzed. Consensus nucleotide sequences of pol and env genes were obtained using NGS. HIV-1 drug resistance analysis was conducted using the Stanford University HIVdb database. CXCR4 cell tropism was predicted using an empirical rule classifier. A protocol for NGS of HIV-1 pol and env genes was developed. The most common HIV-1 surveillance mutations were in the reverse transcriptase. High levels of resistance were observed to non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) in treatment-experienced patients and to NNRTIs in treatment-naïve patients. Low levels of resistance were observed to protease and integrase strand transfer inhibitors (INSTIs). CXCR4 cell tropism was extremely rare. NGS allows for the simultaneous processing of large data sets during epidemiological studies. The introduction of NGS-based protocols allows for performing ART efficiency and tropism monitoring at scale. Full article

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, a burden further exacerbated by HIV co-infection and the increasing prevalence of drug-resistant strains. Although a wide range of laboratory diagnostic methods are currently available, their applicability, implementation, and clinical impact vary substantially across healthcare settings with different levels of complexity and resources. This review provides a comprehensive overview of the main laboratory diagnostic methods for active and latent TB, emphasizing their clinical applicability, implementation challenges, and role within integrated diagnostic strategies. Conventional approaches, such as smear microscopy and culture, are discussed alongside modern diagnostic technologies, including automated nucleic acid amplification tests (NAATs), loop-mediated isothermal amplification (LAMP), line probe assays (LPAs), next-generation sequencing (NGS), and lateral flow assays, highlighting their strengths and limitations in distinct epidemiological and operational contexts. Unlike existing WHO guidelines and prior reviews that predominantly focus on test performance and recommendation status, this review adopts an implementation-oriented perspective, critically examining diagnostic methods in light of real-world constraints, regional disparities, and evidence gaps. Particular attention is given to limitations related to laboratory infrastructure, biosafety, workforce capacity, and sustainability, as well as to under-addressed areas such as latent TB, metagenomic approaches, and the investigation of co-pathogens. By integrating WHO guidance with contextual and operational considerations, this review aims to support rational test selection and the development of flexible, integrated diagnostic workflows tailored to local health system capacity, patient populations, and clinical scenarios, thereby strengthening the effectiveness and equity of TB diagnostic strategies. Full article

Objectives: Respiratory syncytial virus (RSV) is a leading cause of hospitalization for acute lower respiratory tract infections (ALRIs) in children, with bacterial coinfection complicating diagnosis and often driving antibiotic overuse. This study aimed to develop and validate a clinical prediction model using common laboratory biomarkers to enable early, accurate identification of clinically significant bacterial coinfection in children with RSV infection. Methods: A single-center, retrospective cohort study was conducted at Fujian Children’s Hospital, enrolling 518 hospitalized children with RSV infection, which was confirmed via targeted next-generation sequencing (tNGS). Patients were randomly divided into a training set (n = 363) and a test set (n = 155) at a 7:3 ratio. The primary outcome, bacterial coinfection, was defined by a composite reference standard integrating etiological evidence from tNGS with clinical, inflammatory, and imaging data, and adjudicated by a blinded expert panel. LASSO regression identified independent predictors, followed by multivariable logistic regression modeling. Model performance was assessed via discrimination (AUC), calibration (Hosmer–Lemeshow test), and clinical utility (Decision Curve Analysis) in both sets. Results: Neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), and serum amyloid A (SAA) were selected as predictors. The model achieved an AUC of 0.832 (95% CI: 0.788–0.875) in the training set and 0.811 (95% CI: 0.737–0.885) in the test set, with well-calibrated predictions (p > 0.05). Decision curve analysis demonstrated net clinical benefit across 10–80% threshold probabilities. A nomogram was developed for practical application. Conclusions: This study established a model integrating NLR, CRP, and SAA. It offers a reliable tool for the early detection of bacterial coinfection in RSV-infected children, enabling targeted antibiotic stewardship and improving clinical outcomes. Full article

Circulating tumor cells (CTCs) are shed from the primary tumor into the bloodstream and represent dynamic molecular biomarkers for monitoring the progression of cancer. While profiling tumor tissues with over expression of cell surface markers, such as PD-L1 or HER2, is standard in guiding therapy, tissue samples are often inaccessible and inadequate, especially post-surgery or in cases of recurrence. Emerging clinical evidence indicates that CTC counts and biomarker surface expression can predict prognosis and therapeutic resistance more accurately than imaging or tissue-based approaches. Recent advancements in the CTC detection methods, based on physical properties or surface markers (e.g., EpCAM), coupled with next-generation sequencing (NGS) have enabled the isolation of these rare cells and their molecular characterization. Consequently, CTCs provide a real-time alternative, enabling repeated, longitudinal assessment of tumor phenotype and therapeutic response. This review emphasizes the translational potential of surface protein biomarkers on CTCs for profiling, namely PD-L1, HER2, and EGFR, as a clinically actionable approach to stratify patients, guide immunotherapy decisions, and monitor minimal residual disease (MRD), especially when longitudinal tissue biopsies are not feasible. Full article

Introduction: Epilepsy syndromes show marked clinical and genetic heterogeneity, with numerous functionally diverse genes involved in their etiology. Next-generation sequencing (NGS) has facilitated the identification of many monogenic epilepsy syndromes and enables earlier, more accurate diagnosis in pediatric patients. Materials and Methods: This study analyzes the molecular profiles of 87 pediatric patients with various forms of epilepsy in whom pathogenic or likely pathogenic variants were identified. Next-generation sequencing (NGS) using multi-gene epilepsy panels or whole-exome sequencing (WES) was performed. Results: A total of 88 pathogenic or likely pathogenic variants were detected in 48 epilepsy-related genes; 30 variants occurred de novo. SCN1A and KCNQ2 were the most frequent contributors (12.6% and 9.2%, respectively). The highest percentage of positive diagnoses (48%) was observed in patients with developmental and epileptic encephalopathy (DEE), with variants identified in genes including ALG13, ATP1A2, CACNA1A, CDKL5, CHD2, GABRG2, ITPA, KCNQ2, PCDH19, SCN1A, SCN2A, SCN3A, SCN8A, SMC1A, SPTAN1, STXBP1, and UBA5. Pathogenic variants in ANKRD11 were found in four patients with KBG syndrome, while other genes appeared sporadically. Conclusions: Targeted massively parallel sequencing is an effective diagnostic tool for pediatric epilepsy. The presence of numerous single-case findings highlights the high genetic heterogeneity of epilepsy. This approach enabled more precise diagnoses that would not have been achieved through clinical evaluation alone, underscoring the importance of genetic testing for prognosis and treatment planning in pediatric patients with unexplained epilepsy. Full article

Background/Objectives: Cardiac arrhythmias are among the leading causes of sudden cardiac death (SCD). Pathogenic variants in potassium channel genes play a key role in inherited arrhythmia syndromes, yet their contribution in Central Asian populations remains poorly characterized. Methods: We performed targeted next-generation sequencing (NGS) using a 96-gene custom Haloplex panel in 79 Kazakhstani patients with clinically diagnosed arrhythmias, including atrioventricular block, sick sinus syndrome, and atrial fibrillation. Detected variants in potassium channel genes were classified according to ACMG guidelines and correlated with clinical phenotypes. Results: A total of 52 variants were identified across 11 potassium channel genes. Two likely pathogenic variants (KCNH2 p.Cys66Gly and p.Arg176Trp) and six variants of uncertain significance (VUS) in KCNQ1, KCNE2, KCNE3, and KCNJ8 were detected. Two novel previously unreported variants were found in KCNE5 and KCND3. Patients harboring pathogenic variants commonly presented with early-onset arrhythmias or a positive family history of cardiovascular disease. Carriers of KCNH2 variants exhibited mild QT prolongation and recurrent syncope. Conclusions: This is the first genetic study of potassium channel gene mutations in Kazakhstani patients with cardiac arrhythmias. The detection of pathogenic and novel variants highlights the clinical utility of integrating genetic testing into diagnostic and management pathways for arrhythmia syndromes. Population-specific genomic data are essential for improving risk stratification, guiding medication safety, and enabling cascade family screening in Central Asia. Full article

Background/Objectives: In severe peripheral artery disease (PAD) with limb ischemia, hypercholesterolemia (HC) is associated with impaired neovascularization. Extracellular vesicles (EVs) are present within ischemic muscles, and they contain microRNAs (miRs) involved in several biological functions, including angiogenesis and neovascularization. Methods: We used a mouse model of PAD and compared the response to hindlimb ischemia in hypercholesterolemic ApoE^−/− vs. normocholesterolemic mice. Next-generation sequencing (NGS) was used to perform full miR expression profiling in ischemic skeletal muscles and in EVs of varying sizes—large EVs (lEVs) and small EVs (sEVs)—within these muscles. Results: We identified several miRs with potential pro-angiogenic effects (angiomiRs) that are reduced by HC in lEVs (Let-7b-5p, miR-151-3p, Let-7c-5p) or sEVs (miR-21a-5p, miR-196b-5p, miR-340-5p). As proof of principle, we showed that the overexpression of Let-7b-5p in lEVs, or miR-21a-5p in sEVs, can significantly increase the angiogenic capacity of these EVs in vitro. HC also impaired the enrichment of specific angiomiRs in lEVs (miR-100-5p), sEVs (miR-142a-3p), or in both lEVs and sEVs (miR-146b-5p). In silico approaches, including the prediction of miR targets, pathway unions, and gene unions, identified the resulting predictive effects of HC-modulated miRs in EVs on processes with key roles in the modulation of angiogenesis and neovascularization, such as the regulation of the actin cytoskeleton and focal adhesion and the HIF-1, MAPK, AMPK, and PI3K-Akt signaling pathways. Conclusions: Our results constitute an important first step towards the identification of specific miRs that could be targeted to improve EV angiogenic function in hypercholesterolemic conditions and reduce tissue ischemia in patients with severe PAD. Full article

Background: Thymic epithelial tumors (TETs), including thymic carcinomas and thymomas, are rare malignancies originating in the mediastinum. Therapeutic options remain limited for patients experiencing disease progression following platinum-based chemotherapy. High tumor mutational burden (TMB) is uncommon in thymic malignancies but may predict response to immunotherapy. We report a patient with TMB-high TET who participated in the KOSMOS-II study in South Korea and achieved a durable response to atezolizumab without developing immune-related adverse events (irAEs). Case presentation: A 73-year-old woman who had been treated for thymoma 20 years ago presented with a left neck mass. A biopsy of the neck mass confirmed recurrent thymoma, type B3, and her disease progressed despite platinum-based chemotherapy and subsequent pemetrexed treatment. TMB-high thymoma is very rare, but based on the next-generation sequencing (NGS) results, she was diagnosed with TMB-high (20.3 mutations/Mb) thymoma. As TMB-based immunotherapy is not approved in Korea, she was enrolled in the KOSMOS-II study and initiated on atezolizumab following molecular tumor board review. She achieved stable disease after three cycles and has remained progression-free for 14 months, completing 20 cycles without significant irAEs. Notably, her underlying myasthenia gravis did not worsen during treatment. Conclusions: This case demonstrates a favorable outcome with biomarker-directed ICI treatment in recurrent thymoma with limited treatment options, highlighting the importance of appropriate molecular markers to predict drug response. Although TMB-based immunotherapy is FDA-approved in the U.S., it remains unavailable in Korea, underscoring the need to explore flexible access pathways, including the potential use of immunotherapy beyond current indications, to improve treatment options for patients with life-threatening conditions. Full article

Background and Objectives: Aneuploidy is the leading cause of implantation failure and miscarriage, with prevalence increasing with maternal age. Embryos classified as chaotic, characterized by the presence of five or more chromosomal abnormalities, and those with complex aneuploidies, defined by two to four abnormalities, represent a controversial category in preimplantation genetic testing for aneuploidy (PGT-A), as the potential for misclassification remains a significant concern. Materials and Methods: We performed a retrospective study at the Calla IVF Center, Oradea, analyzing 230 blastocysts grouped by maternal age (25–30, 31–35, 36–40, and 41–50 years). A trophoblast biopsy was performed on days 5–7, and the samples were analyzed by next-generation sequencing (NGS). Embryos were classified as euploid, aneuploid, mosaic, or chaotic. The 19 embryos initially diagnosed as chaotic were thawed and subjected to re-biopsy. Statistical analysis included descriptive statistics (chi-square tests and ANOVA) and multivariable regression models, with p < 0.05 as the criterion for statistical significance. Results: Aneuploidy increased with maternal age, from 29.6% in women aged 25–30 years to 68.7% in those aged 41–50 (p = 0.002). Poor-quality blastocysts exhibited higher aneuploidy rates (72.4%) than good-quality embryos (34.6%; p = 0.004). Chaotic embryos comprised 8.3% of the cohort. Upon re-biopsy, none were confirmed as euploid; all remained abnormal and were reassigned to aneuploid, mosaic, or persistently chaotic categories. This finding suggests that apparent euploid results reported elsewhere may reflect technical variability and sampling limitations in PGT-A rather than accurate chromosomal normalization. Conclusions: The prevalence of aneuploid embryos showed a progressive increase with advancing maternal age. Chaotic embryos are heterogeneous, and re-biopsy may help refine the interpretation of complex PGT-A profiles, supporting its role as a diagnostic and quality control tool rather than a strategy to identify euploid embryos. Our study offers novel insights through age-stratified analysis, the integration of morphology with genetics in a Romanian IVF cohort, and a detailed evaluation of chaotic embryos, providing clinical recommendations for patient counseling and embryo selection. Full article

Objectives: To investigate the prognostic value of CEBPA (CCAAT/enhancer-binding protein α) molecular features, such as variant allele frequency (VAF), in patients with de novo acute myeloid leukemia (AML). Methods: Next-generation sequencing (NGS) was used to detect CEBPA mutations in 162 patients with newly diagnosed AML (except acute promyelocytic leukemia). Results: We established 44.2% as the optimal threshold for both maximum VAF and bZIP-domain VAF. The high-VAF group showed higher leukemia burden and inferior event-free survival (EFS). bZIP-domain VAF demonstrated superior prognostic value over maximum VAF (HR: 3.174 vs. 2.460) and was validated across subgroups, namely cytogenetically normal acute myeloid leukemia (CN-AML), chemotherapy-only, and low/intermediate-risk subgroups. Multivariate analysis confirmed high bZIP-domain VAF and DNMT3A mutation as independent risk factors. Conclusions: Our results confirm that the bZIP-domain VAF of CEBPA mutations is a more effective predictor of relapse than the maximum VAF, offering a valuable tool for the early identification of patients at high risk of relapse. Full article

Background: With extensive research and development in the past decade, the affordability of Poly (ADP-ribose) polymerase (PARP) inhibitor therapy has drastically improved. Homologous recombination deficiency (HRD), a key biomarker, has been identified as an important guiding factor for PARP inhibitor therapeutic decisions in breast and ovarian cancer. However, identification of patients who will respond to Poly (ADP-ribose) polymerase (PARP) inhibitor therapy is challenging due to the lack of a unifying morphological phenotype. Current HRD testing via next-generation sequencing (NGS) is tissue-dependent, has high failure rates, misses relevant HRD genes, and involves longer turn-around times. Methods: To overcome these limitations, we developed a multimodal AI model, TRINITY, combining imaging, image-based transcriptome data, and clinico-molecular data, to examine whole-slide images (WSIs) obtained from hematoxylin and eosin (H&E)-stained samples to non-invasively predict HRD status. Results: The TRINITY model, tested on 316 TCGA breast and OV samples, presented a sensitivity of 0.77 and 0.91, NPV of 0.94 and 0.86, PPV of 0.63 and 0.58, specificity of 0.89 and 0.47, and AUC-ROC of 0.91 and 0.72, respectively. The model also yielded a similar outcome in a blind study of 74 samples, with a sensitivity of 81.2, NPV of 0.85, PPV of 0.77, specificity of 0.81, and high AUC-ROC value of 0.89, showing its promising preliminary evidence of predicting HRD status on external cohorts. Conclusions: These findings demonstrate TRINITY’s potential as a rapid, cost-effective, and tissue-sparing alternative to conventional NGS testing. While promising, further validation is needed to establish its generalizability across broader cancer types. Full article

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has had major social and economic consequences worldwide. Whole genome sequencing (WGS) is essential for genomic monitoring, enabling tracking of viral evolution, detection of emerging variants, and identification of introductions and transmission chains to inform timely public health responses. Here, we compile and harmonize SARS-CoV-2 genomic data generated by multiple laboratories across Kazakhstan together with publicly available sequences to provide a national overview of genomic dynamics across successive epidemic waves from 2020 to 2022. We analyzed 4462 genomes deposited in GISAID (including 340 generated in this study), of which 3299 passed Nextclade quality filters, and summarized lineage turnover across major phases (pre-VOC, Alpha, Delta, Omicron BA.1/BA.2, Omicron BA.4/BA.5, and a later recombinant-dominant period). Sequencing intensity varied markedly over time (0.60‰ of confirmed cases during Delta vs. 11.57‰ during the Omicron BA.5 wave), suggesting that lineage diversity and persistence may be underestimated. Pre-VOC circulation included ≥12 Pango lineages with evidence of multiple introductions and sustained local transmission, including a Kazakhstan-restricted B.4.1 lineage that emerged in Nur-Sultan/Astana and disappeared after April 2020. The Tengizchevroil oilfield outbreak comprised B.1.1 viruses with phylogenetic support for ≥three independent introductions. Alpha and Omicron waves were characterized by repeated introductions and heterogeneous origins, whereas Delta was dominated by AY.122 with an additional distinct AY.122 cluster; a notable BF.7 local transmission event was observed during BA.5. We also highlight locally enriched non-lineage-defining mutations. Overall, recurrent importations and variable local amplification shaped SARS-CoV-2 dynamics in Kazakhstan, while interpretation is constrained by strongly time-skewed sequencing. Full article

Identifying pathogens causing periprosthetic joint infection (PJI) is a challenge for clinicians. We aimed to evaluate the application of metagenomic next-generation sequencing (mNGS) to identify pathogens in PJI. A prospective analysis was conducted of patients diagnosed PJI between 2022 and 2024 at twelve hospitals in Taiwan. Both conventional bacterial culture (CMT) and mNGS of joint fluid and debrided tissue were performed. Demographic characteristics, laboratory results and clinical outcomes were collected. The diagnostic performance of these two methods was analyzed. A total of 42 patients with a mean age of 67.9 years were enrolled in analysis. The knee was the most common joint involved (69.1%). A high proportion of patients (78.6%) received prior antibiotics within the two weeks at sample collection. mNGS identified pathogens in 28 out of 42 patients (66.7%), whereas CMT yielded positive results in 12 out of 42 patients (28.6%) (McNemar’s test, p = 0.01). Staphylococcus species was the most common genus detected (n = 11), followed by Cutibacterium (n = 4). Other detected genera included Escherichia, Mycobacterium, Enterobacter, Klebsiella (n = 2 each), Acinetobacter, and Corynebacterium (n = 1 each). Our results support the idea that mNGS could serve as a valuable diagnostic tool for PJI in addition to traditional culture methods. Full article