BACKGROUND: Scrub typhus is a naturally occurring acute infectious disease that is primarily transmitted through the bites of chiggers or larval mites infected by Orientia tsutsugamushi (O. tsutsugamushi). Omadacycline, a novel tetracycline, exhibits potent antibacterial efficacy against both typical bacteria and atypical pathogens. However, omadacycline application in the treatment of scrub typhus remains limited.
METHODS: In the present work, we report several cases of scrub typhus, with the main clinical symptoms being fever, the formation of eschars or ulcers, local or systemic lymphadenopathy, headache, myalgia and rash. Blood samples were collected before omadacycline was administered, and O. tsutsugamushi infection was confirmed through targeted next-generation sequencing (tNGS). After two days of treatment, the patients\' symptoms, including fever, were alleviated, with no adverse drug reactions.
CONCLUSIONS: tNGS is an effective method for diagnosing scrub typhus. Omadacycline can be considered an alternative option for antiinfective therapy in patients with O. tsutsugamushi infections.

We investigated the performance of the targeted next-generation sequencing (tNGS)-based Oxford Nanopore Diagnostics AmPORE TB assay, recently approved by the World Health Organization (WHO) as tuberculosis (TB) diagnostic test for the detection of drug resistance on respiratory specimens. A total of 104 DNA samples from Xpert MTB/RIF-positive TB sputum specimens were tested using the AmPORE TB kit, with the GenoScreen Deeplex Myc-TB as a comparative tNGS assay. For AmPORE TB, DNA samples were divided into five sequencing runs on the MinION device. Data analysis was performed using proprietary software. The WHO catalog of mutations was used for drug resistance interpretation. The assay achieved a high validity rate of 98% (102/104 DNA samples), homogeneous mean reads coverage across TB-positive specimens, and 100% positive and negative agreements for detecting mutations associated with resistance to rifampicin, pyrazinamide, fluoroquinolones, ethambutol, and capreomycin compared with Deeplex Myc-TB. The main discrepancies for the remaining drugs were attributable to the different assay panel designs. The AmPORE TB turnaround time was approximately 5-6 hours from extracted DNA to tNGS reporting for batches of 22 DNA samples. The AmPORE TB assay drastically reduced the time to tNGS reporting from days to hours and showed good performance for drug-resistant TB profiling compared with Deeplex Myc-TB.
OBJECTIVE: Targeted next-generation sequencing (tNGS) of Mycobacterium tuberculosis provides comprehensive resistance predictions matched to new multidrug-resistant/rifampicin-resistant tuberculosis regimens and received World Health Organization approval for clinical use in respiratory samples in 2024. The advanced version of the Oxford Nanopore Diagnostics AmPORE TB tNGS kit was evaluated in this study for the first time and demonstrated good performance, flexibility, and faster turnaround time compared with the existing solutions.

BACKGROUND: Shotgun metagenomic next-generation sequencing (mNGS) is widely used to detect pathogens in bronchoalveolar lavage fluid (BALF). However, mNGS is complex and expensive. This study explored the feasibility of targeted next-generation sequencing (tNGS) in distinguishing lower respiratory tract infections in clinical practice.
METHODS: We used 229 retrospective BALF samples to establish thresholds and diagnostic values in a prospective cohort of 251 patients. After target pathogen selection, primer and probe design, optimization experiments, and bioinformatics analysis, multiplex PCR-based tNGS (mp-tNGS) and hybrid capture-based tNGS (hc-tNGS), targeting 198 and 3060 pathogens (DNA and RNA co-detection workflow) were established and performed.
RESULTS: mp-tNGS and hc-tNGS took 10.3 and 16 h, respectively, with low sequencing data sizes of 0.1 M and 1 M reads, and test costs reduced to a quarter and half of mNGS. The LoDs of mp-tNGS and hc-tNGS were 50-450 CFU/mL. mp-tNGS and hc-tNGS were highly accurate, with 86.5% and 87.3% (vs. 85.5% for mNGS) sensitivities and 90.0% and 88.0% (vs. 92.1% for mNGS) specificities. tNGS detection rates for casual pathogens were 84.3% and 89.5% (vs. 88.5% for mNGS), significantly higher than conventional microbiological tests (P < 0.001). In seven samples, tNGS detected Pneumocystis jirovecii, a fungus not detected by mNGS. Whereas mNGS detected six samples with filamentous fungi (Rhizopus oryzae, Aureobasidium pullulans, Aspergillus niger complex, etc.) which missed by tNGS. The anaerobic bacteria as pathogen in eight samples was failed to detect by mp-tNGS.
CONCLUSIONS: tNGS may offer a new, broad-spectrum, rapid, accurate and cost-effective approach to diagnosing respiratory infections.
BACKGROUND: National Natural Science Foundation of China (81625014 and 82202535).

BACKGROUND: NUP98 rearrangements (NUP98-r) are rare but overrepresented mutations in pediatric acute myeloid leukemia (AML) patients. NUP98-r is often associated with chemotherapy resistance and a particularly poor prognosis. Therefore, characterizing pediatric AML with NUP98-r to identify aberrations is critically important.
METHODS: Here, we retrospectively analyzed the clinicopathological features, genomic and transcriptomic landscapes, treatments, and outcomes of pediatric patients with AML.
RESULTS: Nine patients with NUP98-r mutations were identified in our cohort of 142 patients. Ten mutated genes were detected in patients with NUP98-r. The frequency of FLT3-ITD mutations differed significantly between the groups harboring NUP98-r and those without NUP98-r (P = 0.035). Unsupervised hierarchical clustering via RNA sequencing data from 21 AML patients revealed that NUP98-r samples clustered together, strongly suggesting a distinct subtype. Compared with that in the non-NUP98-r fusion and no fusion groups, CMAHP expression was significantly upregulated in the NUP98-r samples (P < 0.001 and P = 0.001, respectively). Multivariate Cox regression analyses demonstrated that patients harboring NUP98-r (P < 0.001) and WT1 mutations (P = 0.030) had worse relapse-free survival, and patients harboring NUP98-r (P < 0.008) presented lower overall survival.
CONCLUSIONS: These investigations contribute to the understanding of the molecular characteristics, risk stratification, and prognostic evaluation of pediatric AML patients.

OBJECTIVE: Genomic newborn screening programs are emerging worldwide. With the support of the local pediatric team of Liege, Belgium, we developed a panel of 405 genes that are associated with 165 early-onset, treatable diseases with the goal of creating a newborn screening test using targeted next-generation sequencing for all early-onset, treatable, and serious conditions.
METHODS: A process was developed that informed the future parents about the project and collected their consent during a face-to-face discussion with a trained investigator. The first baby was screened on 1 September 2022. The main objective of the study was to test the feasibility and the acceptability of targeted sequencing at birth as a first-tier newborn screening approach to detect treatable genetic conditions or genetic conditions for which a pre-symptomatic or early symptomatic clinical trial is available.
RESULTS: As of 20 June 2024, the parents of 4425 children had been offered the test; 4005 accepted (90.5%) and 420 refused (9.5%). The main reasons for refusal were the research nature of the project and the misunderstanding of what constitutes genetic conditions.
CONCLUSIONS: These data demonstrate the high acceptability of genomic newborn screening in a properly informed population.

Identifying mutations in cancer-associated genes to guide patient treatments is essential for precision medicine. Circulating tumor DNA (ctDNA) offers valuable insights for early cancer detection, treatment assessment, and surveillance. However, a key issue in ctDNA analysis from the bloodstream is the choice of a technique with adequate sensitivity to identify low frequent molecular changes. Next-generation sequencing (NGS) technology, evolving from parallel to long-read capabilities, enhances ctDNA mutation analysis. In the present review, we describe different NGS approaches for identifying ctDNA mutation, discussing challenges to standardized methodologies, cost, specificity, clinical context, and bioinformatics expertise for optimal NGS application.

OBJECTIVE: To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection.
METHODS: We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People\'s Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics.
RESULTS: The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.

Lack of appropriate early diagnostic tools for drug-resistant tuberculosis (DR-TB) and their incomplete drug susceptibility testing (DST) profiling is concerning for TB disease control. Existing methods, such as phenotypic DST (pDST), are time-consuming, while Xpert MTB/RIF (Xpert) and line probe assay (LPA) are limited to detecting resistance to few drugs. Targeted next-generation sequencing (tNGS) has been recently approved by WHO as an alternative approach for rapid and comprehensive DST. We aimed to investigate the performance and feasibility of tNGS for detecting DR-TB directly from clinical samples in Bangladesh. pDST, LPA and tNGS were performed among 264 sputum samples, either rifampicin-resistant (RR) or rifampicin-sensitive (RS) TB cases confirmed by Xpert assay. Resistotypes of tNGS were compared with pDST, LPA and composite reference standard (CRS, resistant if either pDST or LPA showed a resistant result). tNGS results revealed higher sensitivities for rifampicin (RIF) (99.3%), isoniazid (INH) (96.3%), fluoroquinolones (FQs) (94.4%), and aminoglycosides (AMGs) (100%) but comparatively lower for ethambutol (76.6%), streptomycin (68.7%), ethionamide (56.0%) and pyrazinamide (50.7%) when compared with pDST. The sensitivities of tNGS for INH, RIF, FQs and AMGs were 93.0%, 96.6%, 90.9%, and 100%, respectively and the specificities ranged from 91.3 to 100% when compared with CRS. This proof of concept study, conducted in a high-burden setting demonstrated that tNGS is a valuable tool for identifying DR-TB directly from the clinical specimens. Its feasibility in our laboratory suggests potential implementation and moving tNGS from research settings into clinical settings.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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%).
UNASSIGNED: 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.

BACKGROUND: The global prospective surveillance data showed the re-emergence of mycoplasma pneumoniae pneumonia (MPP) in Europe and Asia after the coronavirus disease 2019 pandemic. We sought to observe the effect of macrolide antibiotics in the treatment of MPP carrying a macrolide-resistant mutation gene and the potential of targeted next-generation sequencing (tNGS) as a front-line diagnostic in MPP patients.
METHODS: The baseline characteristics of 91 children with MPP hospitalized from January to October 2023 were retrospectively analyzed. They were divided into two groups according to whether carrying the macrolide-resistant mutation or not. The logistic and linear regression analyses were used to determine whether the mutation was a standalone predictive predictor of the duration of fever and hospital length of stay.
RESULTS: First, no patients had a fever for ≥ 7 days after macrolide treatment. But length of stay and hormone concentration were significantly different between the two groups (P < 0.05). There were also no statistical association between the mutation and the duration of fever and hospital length of stay.
CONCLUSIONS: Macrolides can be administered to MPP children carrying a macrolide-resistant mutation. tNGS can be seen as a front-line diagnostic in MPP.