Relevance of genetic testing in the gene-targeted trial era: the Rostock Parkinson's disease study.

Estimates of the spectrum and frequency of pathogenic variants in Parkinson's disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinson's disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (∼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that ∼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.

Exploring Atrial Fibrillation: Understanding the Complex Relation Between Lifestyle and Genetic Factors.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide across diverse ethnic groups. Among these, atrial fibrillation (AF) stands as one of the most prevalent types of arrhythmias and the primary cause of stroke. Risk factors associated with AF include alcohol consumption, aging, high blood pressure, hypertension, inflammation, and genetic factors. A family history of CVD could indicate an increased risk. Consequently, genetic, and genomic testing should be performed to identify the molecular etiology of CVDs and assess at-risk patients. It is important to note that CVDs are the results of the complex interplay of genes and environmental factors, including ethnicity. In this case, the proband's clinic story includes a history of smoking abuse for 10 years (10 cigarettes per day), obesity, hypertension, and an associated familial history. These risk factors, along with genetic variants, could trigger the early onset of AF. In recent years, genetic and genomic studies have significantly advanced our understanding of CVD etiology, given that next-generation sequencing (NGS) allows for the identification of genetic variants that could contribute to these pathologies. Furthermore, NGS facilitates early diagnosis, personalized pharmacological approaches, and identification of novel biomarkers. Thus, NGS is a valuable tool in CVD management. However, such studies are limited in Ecuador, a low- and middle-income country. Several challenges contribute to this gap, encompassing economic, infrastructural, and educational obstacles. Notably, the cost of genetic and genomic studies may also pose a barrier, restricting access to a portion of the population. In this case report, we present a 56-year-old Ecuadorian woman, who has been diagnosed with AF; however, after performing NGS no disease-associated variants were found, despite having strong clinical signs and symptoms. In summary, this case report contributes valuable insights into the complex interplay between genetic and lifestyle factors in the development and management of AF. The case report aims to underscore the potential impact of genetic variants on disease risk, even when classified as variants of uncertain significance, and the importance of an integral approach to patient care that includes genetic screening, lifestyle interventions, and tailored pharmacological treatment.

Clinical Utility of Plasma Microbial Cell-Free DNA Sequencing Among Immunocompromised Patients With Pneumonia.

Background: Plasma microbial cell-free DNA (mcfDNA) sequencing can establish the etiology of multiple infectious syndromes by identifying microbial DNA in plasma. However, data are needed to define the clinical scenarios where this tool offers the highest clinical benefit. Methods: We conducted a prospective multicenter observational study that evaluated the impact of plasma mcfDNA sequencing compared with usual care testing among adults with hematologic malignancies. This is a secondary analysis of an expanded cohort that evaluated the clinical utility of plasma mcfDNA sequencing across prespecified and adjudicated outcomes. We examined the percentage of participants for whom plasma mcfDNA sequencing identified a probable cause of pneumonia or clinically relevant nonpneumonia infection. We then assessed potential changes in antimicrobial therapy based on plasma mcfDNA sequencing results and the potential for early mcfDNA testing to avoid bronchoscopy and its associated adverse events. Results: Of 223 participants, at least 1 microbial detection by plasma mcfDNA sequencing was adjudicated as a probable cause of pneumonia in 57 (25.6%) and a clinically relevant nonpneumonia infection in 88 (39.5%). A probable cause of pneumonia was exclusively identified by plasma mcfDNA sequencing in 23 (10.3%) participants. Antimicrobial therapy would have changed for 41 (18.4%) participants had plasma mcfDNA results been available in real time. Among the 57 participants with a probable cause of pneumonia identified by plasma mcfDNA sequencing, bronchoscopy identified no additional probable cause of pneumonia in 52 (91.2%). Conclusions: Plasma mcfDNA sequencing could improve management of both pneumonia and other concurrent infections in immunocompromised patients with suspected pneumonia.

The protective effects of aqueous extract of Schisandra sphenanthera against alcoholic liver disease partly through the PI3K-AKT-IKK signaling pathway.

Purpose: This study aimed to investigated the key chemical components and the effect of the aqueous extract of Schisandra sphenanthera (SSAE) on alcoholic liver disease (ALD) and the related molecular mechanism. Methods: This study employed UPLC-Q-TOF-MS/MS to identify the chemical compositions in SSAE. ALD rat model was established through oral administration of white spirit. Transcriptome sequencing, weighted gene co-expression network construction analysis (WGCNA), and network pharmacology were used to predict key compositions and pathways targeted by SSAE for the treatment of ALD. Enzyme-linked immunosorbent assay (ELISA), biochemical kits, hematoxylin-eosin (HE) staining, Western blotting (WB) analysis, and immunohistochemical analysis were used to validate the mechanism of action of SSAE in treating ALD. Results: Active ingredients such as schisandrin A, schisandrol A, and schisandrol B were found to regulate the PI3K/AKT/IKK signaling pathway. Compared to the model group, the SSAE group demonstrated significant improvements in cellular solidification and tissue inflammation in the liver tissues of ALD model rats. Additionally, SSAE regulated the levels of a spartate aminotransferase (AST), alanine aminotransferase (ALT), alcohol dehydrogenase (ADH), and aldehyde Dehydrogenase (ALDH) in serum (P < 0.05); Western blotting and immunohistochemical analyses showed that the expression levels of phosphorylated PI3K, AKT, IKK, NFκB, and FOXO1 proteins were significantly reduced in liver tissues (P < 0.05), whereas the expression level of Bcl-2 proteins was significantly increased (P < 0.05). Conclusion: The active components of SSAE were schisandrin A, schisandrol A, and schisandrol B, which regulated the phosphorylation levels of PI3K, AKT, IKK, and NFκB and the expression of FOXO1 protein and upregulated the expression of Bcl-2 protein in the liver tissues of ALD rats. These findings indicate that SSAE acts against ALD partly through the PI3K-AKT-IKK signaling pathway. This study provided a reference for future research and treatment of ALD and the development of novel natural hepatoprotective drugs.

Pure large-cell neuroendocrine carcinoma of the ovary with a somatic BRCA1 mutation: The first reported case and the review of the literature.

Pure large-cell neuroendocrine carcinomas of the ovary are extremely rare, so there is a lack of molecular information on this type of cancer. Herein, we presented a pure primary large-cell neuroendocrine carcinomas of the ovary in a 72-year-old female with a pathogenic somatic mutation at the c.5332+1g>a splice site of the BRCA1 gene and with no TP53 mutation. She was uneventful 32 months after the operation and chemotherapies. To the best of our knowledge, this is the first report of a BRCA1 somatic mutation in the ovary large-cell neuroendocrine carcinomas. Testing BRCA1/2 mutations in patients with large ovarian cell neuroendocrine carcinomas might provide an opportunity for their future target treatments. It would expand our understanding.

Identifying potential therapeutic targets in lung adenocarcinoma: a multi-omics approach integrating bulk and single-cell RNA sequencing with Mendelian randomization.

Our research aimed to identify new therapeutic targets for Lung adenocarcinoma (LUAD), a major subtype of non-small cell lung cancer known for its low 5-year survival rate of 22%. By employing a comprehensive methodological approach, we analyzed bulk RNA sequencing data from 513 LUAD and 59 non-tumorous tissues, identifying 2,688 differentially expressed genes. Using Mendelian randomization (MR), we identified 74 genes with strong evidence for a causal effect on risk of LUAD. Survival analysis on these genes revealed significant differences in survival rates for 13 of them. Our pathway enrichment analysis highlighted their roles in immune response and cell communication, deepening our understanding. We also utilized single-cell RNA sequencing (scRNA-seq) to uncover cell type-specific gene expression patterns within LUAD, emphasizing the tumor microenvironment's heterogeneity. Pseudotime analysis further assisted in assessing the heterogeneity of tumor cell populations. Additionally, protein-protein interaction (PPI) network analysis was conducted to evaluate the potential druggability of these identified genes. The culmination of our efforts led to the identification of five genes (tier 1) with the most compelling evidence, including SECISBP2L, PRCD, SMAD9, C2orf91, and HSD17B13, and eight genes (tier 2) with convincing evidence for their potential as therapeutic targets.

The role of metabolic memory in diabetic kidney disease: identification of key genes and therapeutic targets.

Diabetic kidney disease (DKD) is characterized by complex pathogenesis and poor prognosis; therefore, an exploration of novel etiological factors may be beneficial. Despite glycemic control, the persistence of transient hyperglycemia still induces vascular complications due to metabolic memory. However, its contribution to DKD remains unclear. Using single-cell RNA sequencing data from the Gene Expression Omnibus (GEO) database, we clustered 12 cell types and employed enrichment analysis and a cell‒cell communication network. Fibrosis, a characteristic of DKD, was found to be associated with metabolic memory. To further identify genes related to metabolic memory and fibrosis in DKD, we combined the above datasets from humans with a rat renal fibrosis model and mouse models of metabolic memory. After overlapping, NDRG1, NR4A1, KCNC4 and ZFP36 were selected. Pharmacology analysis and molecular docking revealed that pioglitazone and resveratrol were possible agents affecting these hub genes. Based on the ex vivo results, NDRG1 was selected for further study. Knockdown of NDRG1 reduced TGF-ß expression in human kidney-2 cells (HK-2 cells). Compared to that in patients who had diabetes for more than 10 years but not DKD, NDRG1 expression in blood samples was upregulated in DKD patients. In summary, NDRG1 is a key gene involved in regulating fibrosis in DKD from a metabolic memory perspective. Bioinformatics analysis combined with experimental validation provided reliable evidence for identifying metabolic memory in DKD patients.

Mutations Associated With High-Grade irAEs in NSCLC Patients Receiving Immunotherapies.

OBJECTIVES: Compared to low-grade irAEs, high-grade irAEs are more often dose-limiting and can alter the long-term treatment options for a patient. Predicting the incidence of high-grade irAEs would help with treatment selection and therapeutic drug monitoring. MATERIALS AND METHODS: We performed a retrospective study of 430 stage III and IV patients with non-small cell lung cancer (NSCLC) who received an immune checkpoint inhibitor (ICI), either with or without chemotherapy, at a single comprehensive cancer center from 2015 to 2022. The study team retrieved sequencing data and complete clinical information, including detailed irAEs medical records. Fisher's exact test was used to determine the association between mutations and the presence or absence of high-grade irAEs. Patients were analyzed separately based on tumor subtypes and sequencing platforms. RESULTS: High-grade and low-grade irAEs occurred in 15.2% and 46.2% of patients, respectively. Respiratory and gastrointestinal irAEs were the 2 most common irAEs. The distribution of patients with or without irAEs was similar between ICI and ICI+chemotherapy-treated patients. By analyzing the mutation data, we identified 5 genes (MYC, TEK, FANCA, FAM123B, and MET) with mutations that were correlated with an increased risk of high-grade irAEs. For the adenocarcinoma subtype, mutations in TEK, MYC, FGF19, RET, and MET were associated with high-grade irAEs; while for the squamous subtype, ERBB2 mutations were associated with high-grade irAEs. CONCLUSION: This study is the first to demonstrate that specific tumor mutations correlate with the incidence of high-grade irAEs in patients with NSCLC treated with an ICI, providing molecular guidance for treatment selection and drug monitoring.

Concepts and new developments in droplet-based single cell multi-omics.

Single cell sequencing technologies have become a fixture in the molecular profiling of cells due to their ease, flexibility, and commercial availability. In particular, partitioning individual cells inside oil droplets via microfluidic reactions enables transcriptomic or multi-omic measurements for thousands of cells in parallel. Complementing the multitude of biological discoveries from genomics analyses, the past decade has brought new capabilities from assay baselines to enable a deeper understanding of the complex data from single cell multi-omics. Here, we highlight four innovations that have improved the reliability and understanding of droplet microfluidic assays. We emphasize new developments that further orient principles of technology development and guidelines for the design, benchmarking, and implementation of new droplet-based methodologies.

Spatial Variation of the Gastrointestinal Microbiota in Response to Long-Term Administration of Vonoprazan in Mice With High Risk of Gastric Cancer.

BACKGROUND: Vonoprazan, a potassium-competitive acid blocker, is superior to traditional proton pump inhibitor (PPI) in acid suppression and has been approved in the treatment of acid-related disorders. Accumulating evidence suggest associations between PPI use and gut microbiota, yet the effect of vonoprazan on GI microbiota is obscure. METHODS: Transgenic FVB/N insulin-gastrin (INS-GAS) mice as a model of gastric cancer (GC) were administered vonoprazan by gavage every other day for 12 weeks. Stomachs were evaluated by histopathology, Ki-67 proliferation index, and inflammatory cytokines. The mucosal and lumen microbiota from stomach, jejunum, ileum, cecum, and feces were detected using 16S rRNA gene sequencing. RESULTS: Higher incidence of intestinal metaplasia and epithelial proliferation were observed in the vonoprazan group than that in the control mice. Vonoprazan also elevated the gastric expression of proinflammatory cytokines, including TNF-α, IL-1ß, and IL-6. Each mice comprised a unique microbiota composition that was consistent across different niches. The structure of GI microbiota changed dramatically after vonoprazan treatment with the stomach being the most disturbed segment. Vonoprazan administration shifted the gut microbiota toward the enrichment of pathogenic Streptococcus, Staphylococcus, Bilophila, and the loss of commensal Prevotella, Bifidobacterium, and Faecalibacterium. Interestingly, compared to the controls, microbial interactions were weaker in the stomach while stronger in the jejunum of the vonoprazan group. CONCLUSIONS: Long-term vonoprazan treatment promoted gastric lesions in male INS-GAS mice, with the disequilibrium of GI microbiome. The clinical application of vonoprazan needs to be judicious particularly among those with high risk of GC.

How does orthodontic tooth movement influence the dental pulp? RNA-sequencing on human premolars.

OBJECTIVES: The objective of this study is to analyse the gene expression profile of the dental pulp (DP) of human premolars subjected to 7 and 28 days of orthodontic force (OF) in vivo by using RNA sequencing. The maxillary and mandibular DP were additionally compared. METHODS: Healthy patients requiring orthodontic premolar extractions were randomly assigned to one of the three groups: control (CG) where no OF was applied, 7 and 28 days, where premolars were extracted either 7 or 28 days after the application of a 50-100 g OF. Total RNA was extracted from the DP and analysed via RNA-seq. Differentially expressed genes (DEGs) were identified using a false discovery rate and fold change threshold of <0.05 and ≥1.5, respectively. Functional analysis was performed. RESULTS: After 7 days of OF, pulp reaction indicates immune response, hypoxia, DNA damage and epigenetic regulation. After 28 days, cell adhesion, migration, organization and tissue repair are evident. The maxillary and mandibular pulp tissues react differently to OF. The maxilla exhibits minimal alterations, mostly related to immune response at 7 days and tissue repair at 28 days, whereas the mandible shows mostly DNA damage and epigenetic regulation at 7 days and return to the original state at 28 days. CONCLUSIONS: This study demonstrates that the early reaction of the DP to OF is marked by immune response, hypoxia and DNA damage. In contrast, after 28 days, cell adhesion, migration, organization, tissue repair and dentine formation are observed. Maxillary and mandibular premolars react differently to OF: although the maxilla exhibits minimal alterations at both time points, the mandible mostly shows DNA damage, epigenetic regulation, and immune response at 7 days. These disparities could stem from different blood supplies or the lower maxillary bone density, potentially triggering faster biological changes. Our findings provide insights into the gene regulatory networks modulating DP response to OF.

Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on short tandem repeat sequence nomenclature.

The DNA Commission of the International Society for Forensic Genetics (ISFG) has developed a set of nomenclature recommendations for short tandem repeat (STR) sequences. These recommendations follow the 2016 considerations of the DNA Commission of the ISFG, incorporating the knowledge gained through research and population studies in the intervening years. While maintaining a focus on backward compatibility with the CE data that currently populate national DNA databases, this report also looks to the future with the establishment of recommended minimum sequence reporting ranges to facilitate interlaboratory comparisons, automated solutions for sequence-based allele designations, a suite of resources to support bioinformatic development, guidance for characterizing new STR loci, and considerations for incorporating STR sequences and other new markers into investigative databases.

Identification of copy-number variants in patients with overgrowth disorders.

Overgrowth syndromes (OGS) comprise a heterogeneous group of disorders whose main characteristic is that the weight, height or the head circumference are above the 97th centile or 2-3 standard deviations above the mean for age, gender, and ethnic group. Several copy-number variants (CNVs) have been associated with the development of OGS, such as the 5q35 microdeletion or the duplication of the 15q26.1-qter, among many others. In this study, we have applied 850K SNP-arrays to 112 patients and relatives with OGS from the Spanish OverGrowth Registry Initiative. We have identified CNVs associated with the disorder in nine individuals (8%). Subsequently, whole genome sequencing (WGS) analysis was performed in these nine samples in order to better understand these genomic imbalances. All the CNVs were detected by both techniques, settling that WGS is a useful tool for CNV detection. We have found six patients with genomic abnormalities associated with previously well-established disorders and three patients with CNVs of unknown significance, which may be related to OGS, based on scientific literature. In this report, we describe these findings and comment on genes associated with OGS that are located within the CNV regions.

Molecular analysis of 31 cases with fetal skeletal dysplasia.

OBJECTIVES: The aim of this study was to describe the prenatal ultrasound findings of fetuses with skeletal dysplasia and to evaluate the genetic variations by molecular genetic analysis. METHODS: Between August 1, 2018 and March 1, 2023, we conducted a retrospective case series at a tertiary referral center involving patients with fetal skeletal abnormalities. For cases referred for a possible diagnosis of fetal skeletal dysplasia, an ultrasound database and prenatal genetic counseling records were first searched. Terminated cases diagnosed with skeletal dysplasia by pathologic and radiologic findings and cases with skeletal dysplasia proven by postnatal clinical findings were included in the study. RESULTS: Between 2018 and 2023, a total of 64 cases were diagnosed as skeletal dysplasia based on radiologic findings, pathologic findings, and clinical features. The median week of the first ultrasound performed on patients is 19 0/7 weeks, while the median week of the ultrasound in which skeletal dysplasia is suspected is 21 3/7 weeks. Although micromelia was evaluated as a common feature in all cases, the most common concomitant anomaly was thoracic hypoplasia. Exome sequencing analysis was achieved in 31 (48 %) of cases. In 31 cases, in total of 35 pathogenic single gene mutations and 5 VUS (variants of uncertain significance) variants composing of 23 autosomal dominant, 10 autosomal recessive and 2 X linked recessive mutations were determined. CONCLUSIONS: Prenatal ultrasound findings can lead us to specific diagnoses, and with the appropriate molecular analysis method, a definitive diagnosis can be made without wasting time and money.

Eight new variants of HLA-DQB1*03 identified by next-generation sequencing.

Genomic sequence of HLA-DQB1*03:01:01:60, -DQB1*03:01:01:61, -DQB1*03:01:01:62, -DQB1*03:01:01:63, -DQB1*03:02:01:23, -DQB1*03:02:01:24, -DQB1*03:02:01:25 and -DQB1*03:03:02:14 alleles in Spanish individuals.

Characterisation of the novel HLA-DRB1*08:126 allele by sequencing-based typing.

HLA-DRB1*08:126 differs from HLA-DRB1*08:04:01:01 by one nucleotide substitution in codon 152 in exon 3.

Identification of the novel HLA-DPB1*14:01:15 allele in a Brazilian bone marrow donor.

The novel HLA-DPB1*14:01:15 allele differs from DPB1*14:01:01:01 by change of C > T in exon 3.

Quantification using a HPLC-FLD-based Detector and Molecular Identifi-cation of Ochratoxin-A Producing Aspergillus Species Isolated from Stored Grain Samples.

AIM: Quantification using an HPLC-FLD based detector and Molecular identification of Ochratoxin-A producing Aspergillus Species isolated from stored grain samples. BACKGROUND: Fungi are cosmopolitan in origin and are known to grow in any suitable substra-tum. In the present investigation, Aspergillus species isolated from stored grain samples were analyzed for ochratoxin-A production. OBJECTIVE: The objective of this study is the quantification of Ochratoxin and identification of ochratoxigenic fungi. METHODS: A total of n=34 black Aspergilli and n=1 Ochre Aspergilli were isolated from grain samples of Bihar, India and it was tested for OTA production. The limit of detection (LOD) is found to be 0.33µg/Kg and the limit of quantification (LOQ) is found to be 1µg/Kg for OTA in HPLC-FLD. RESULTS: In the present study, out of all the fungal isolates, only TiB fungal isolate was able to produce the ochratoxin-A above the level of LOQ. The positive isolate TiB obtained from stored sesame seed samples was able to produce 25.54 µg/Kg of OTA. ITS sequence analysis of TiB isolate was able to matche 100% with Aspergillus welwitschiae and Aspergillus foetidus. CONCLUSION: This is the initial report of ochratoxigenic Aspergillus fungal isolate isolated from stored sesame seed samples of flood-prone areas of Bihar, India.

Bronchial lavage tNGS in the diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis, remains a significant global health concern. Targeted Next-Generation Sequencing (tNGS) has emerged as a rapid and comprehensive diagnostic tool for tuberculosis, offering advantages over traditional methods and serving as an effective alternative for drug susceptibility testing and the detection of drug-resistant tuberculosis. OBJECTIVE: This study aimed to retrospectively analyze the clinical characteristics of pulmonary tuberculosis patients. After explore the application value of targeted next-generation sequencing technology in this patient population, providing valuable insights for clinical diagnosis and treatment. METHODS: In this retrospective study, we analyzed data from 65 patients with laboratory-confirmed tuberculosis admitted to Tianjin Baodi Hospital from November 14, 2020, to February 1, 2023. Patients underwent bronchoalveolar lavage fluid (BALF) testing, including acid-fast staining, culture, and tNGS. Biopsies and histopathological examinations were performed on some patients, along with comprehensive radiological assessments for all. RESULTS: Among the 65 pulmonary tuberculosis patients, targeted next-generation sequencing detected pathogens in bronchoalveolar lavage fluid with a positivity rate of 93.8%, significantly higher than traditional methods such as acid-fast staining, culture, and pathology. Compared to bronchoalveolar lavage fluid smear, targeted next-generation sequencing demonstrated significantly higher diagnostic sensitivity (98.46% vs. 26.15%) and accuracy (98.46% vs. 26.15%). CONCLUSION: Targeted next-generation sequencing, with its high sensitivity and specificity compared to traditional methods, provides unique advantages in detecting pathogens among these patients, highlighting its importance in disease management.