[Mutation spectrum analysis of 23-site chip neonatal deafness genetic screening].

Objective:To analyze the mutation spectrum of 23-site chip newborn deafness genetic screening in Beijing, and to provide basis for genetic counseling and clinical diagnosis and treatment. Methods:The study included 21 006 babies born in Beijing from December 2022 to June 2023. All subjects underwent newborn deafness genetic screening in Beijing Tongren Hospital, covering 23 variants in 4 genes, the GJB2 gene（c.35delG, c.176_191del16, c.235delC, c.299_300delAT, c.109G>A, c.257C>G, c.512insAACG, c.427C>T, c.35insG）, SLC26A4 gene（c.919-2A>G, c.2168A>G, c.1174A>T, c.1226G>A, c.1229C>T, c.1975G>C, c.2027T>A, c.589G>A, c.1707+5G>A, c.917insG, c.281C>T）, Mt12SrRNA（m.1555A>G, m.1494C>T） and GJB3 gene（c.538C>T）. The mutation detection rate and allele frequency were analyzed. Results:The overall mutation detection rate was 11.516%（2 419/21 006）, with the GJB2 gene being the most frequently involved at 9.097%（1 911/21 006）, followed by the SLC26A4 gene at 2.123%（446/21 006）, the GJB3 gene at 0.362%（76/21 006） and Mt12SrRNA at 0.176%（37/21 006）. Among the GJB2 genes, c.109G>A and c.235delC mutation detection rates were the highest, with 6.579%（1 382/21 006） and 1.795%（377/21 006）, respectively. Of the SLC26A4 genes, c.919-2A>G and c.2168A>G had the highest mutation rates of 1.423%（299/21 006） and 0.233%（49/21 106）, respectively. Regarding the allele frequency, GJB2 c.109G>A was the most common variant with an allele frequency of 3.359%（1 411/42 012）, followed by the GJB2 c.235delC at 0.897%（377/42 012） and the SLC26A4 c.919-2A>G at 0.719%（302/42 012）. Conclusion:23-site chip newborn deafness genetic screening in Beijing showed that GJB2 c.109G>A mutation detection rate and allele frequency were the highest. This study has enriched the epidemiological data of 23-site chip genetic screening mutation profiles for neonatal deafness, which can provide evidence for clinical practice.

Nitrogen-phosphorus dual-doped auricularia auricula porous carbon as host for Li-S battery.

A nitrogen-phosphorus dual-doped porous spore carbon (NP-PSC) positive electrode matrix was prepared using native auricularia auricula as solid medium based on the principle of biomass rot. Yeast was introduce and cultured by the auricularia auricula solid medium. The freeze-drying and carbonization activation processes made the materials present a three-dimensional porous spore carbon aerogel properties. Yeast fermentation transformed auricularia auricula from blocky structure to porous structure and introduced nitrogen-phosphorus dual-doping. The physical and chemical properties of the prepared materials were characterized in detail. Electrochemical performance of NP-PSC in Li-S batteries was systematically investigated. Porous structure and heteroatom-doping improved the electrochemical performance, which is much superior to conventional activated carbon materials.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

An early and trustable indicator suggestive of non-tuberculosis mycobacteria isolation in a high tuberculosis burden setting.

BACKGROUND: Distinguishing between nontuberculous mycobacterial (NTM) lung infections and pulmonary tuberculosis becomes challenging due to their similar clinical manifestations and radiological images. Consequently, instances of delayed diagnosis or misdiagnosis are highly frequent. A feasible and reliable indicator of the existence of NTM in the early stages of the disease would help to solve this dilemma. METHODS: In this study, we evaluated the potential of smear-positive and Xpert assay (Cepheid, USA) negative outcomes as an early indicator of possible NTM infection in a high TB-burden setting retrospectively and prospectively. RESULTS: During the study period, 12·77% (138/1081) of the smear-positive cases yielded negative outcomes with the simultaneous Xpert assay. From the 110 patients who yielded smear-positive/Xpert-negative outcomes and cultivated strain as well, 105 (95·45%) were proved to have NTM isolated. By incorporating an additional criterion of a negative result from the Interferon-gamma release assay, the accuracy of the screening method reached 100%. Regarding the NTM presence prediction value, smear-positive/Xpert-negative has a sensitivity of 24·86% (45/181) in all NTM isolated cases but 93·75-96·55% accuracy in retrospective study or 93·75% accuracy in prospective study in smear-positive NTM isolated cases. In addition, the specificity was ∼99·47% (943/948) in smear-positive tuberculosis cases. CONCLUSION: The clue of the presence of NTM could be obtained on the first day of the hospital visit due to the point of care (POC) feature of smear testing and Xpert assay. About one-fourth of the NTM-isolated patients would benefit from this rapid, convenient, and reliable screening strategy in the given circumstance. Smear-positive/Xpert-negative outcome is an early, trustable indicator that is indicative of NTM isolation.

Aptamer inhibitor selection of SpyCas9 through CE-SELEX.

CRISPR/Cas9 is a natural immune system of archaea and bacteria, which has been widely used in gene editing. In order to better control and improve the accuracy and safety of the system, inhibitors for SpyCas9 as "switches" have been selected for several years. The available inhibitors currently are all natural polypeptides inhibitors derived from phages, except one small molecule inhibitor. These natural inhibitors are challenging to obtain and are available in limited quantities, and the small molecule inhibitor is cytotoxic. Herein, we discover aptamers against the SpyCas9 protein, by coupling CE-SELEX within one-round pressure controllable selection strategy. One of the identified aptamers, Apt2, shows high affinity at the nanomolar level and leads for effective SpyCas9 enzymatic inhibition in vitro. It is predicted that Apt2 interacts with the HNH and RuvC domains of SpyCas9, competitively inhibiting the binding of substrate DNA to SpyCas9. The proposed aptamer inhibitor is the oligonucleotide inhibitor of SpyCas9, which has the potential in construction of the universal, simple and precise CRISPR-Cas9 system activity control strategy. Meanwhile, these aptamers could also be valuable tools for study of the functions of CRISPR/Cas9 and the related functional mechanisms.

Cryogel wound dressings based on natural polysaccharides perfectly adhere to irregular wounds for rapid haemostasis and easy disassembly.

Skin injuries can have unexpected surfaces, leading to uneven wound surfaces and inadequate dressing contact with these irregular surfaces. This can decrease the dressing's haemostatic action and increase the healing period. This study recommends the use of sticky and flexible cryogel coverings to promote faster haemostasis and efficiently handle uneven skin wounds. Alginate cryogels have a fast haemostatic effect and shape flexibility due to their macroporous structure. The material demonstrates potent antibacterial characteristics and enhances skin adherence by adding grafted chitosan with gallic acid. In irregular defect wound models, cryogels can cling closely to uneven damage surfaces due to their amorphous nature. Furthermore, their macroporous structure allows for quick haemostasis by quickly absorbing blood and wound exudate. After giving the dressing a thorough rinse, its adhesive strength reduces and it is simple to remove without causing any damage to the wound. Cryogel demonstrated faster haemostasis than gauze in a wound model on a rat tail, indicating that it has considerable potential for use as a wound dressing in the biomedical area.

SPDEF drives pancreatic adenocarcinoma progression via transcriptional upregulation of S100A16 and activation of the PI3K/AKT signaling pathway.

Pancreatic adenocarcinoma (PAAD) is a notably aggressive malignancy with limited treatment options and an unfavorable prognosis for patients. We aimed to investigate molecular mechanisms by which Sam's pointed domain-containing ETS transcription factor (SPDEF) exerts effects on PAAD progression. We analyzed differentially expressed genes (DEGs) and their integration with ETS family members using the The Cancer Genome Atlas (TCGA) database, hence identifying SPDEF as a core gene in PAAD. Kaplan-Meier survival analysis confirmed SPDEF's prognostic potential. In vitro experiments validated the association with cell proliferation and apoptosis, affecting pancreatic cancer cell dynamics. We detected increased SPDEF expression in PAAD tumor samples. Our in vitro studies revealed that SPDEF regulates mRNA and protein expression levels, and significantly affects cell proliferation. Moreover, SPDEF was associated with reduced apoptosis and enhanced cell migration and invasion. In-depth analysis of SPDEF-targeted genes revealed four crucial genes for advanced prognostic model, among which S100A16 was significantly correlated with SPDEF. Mechanistic analysis showed that SPDEF enhances the transcription of S100A16, which in turn enhances PAAD cell migration, proliferation, and invasion by activating the PI3K/AKT signaling pathway. Our study revealed the critical role of SPDEF in promoting PAAD by upregulating S100A16 transcription and stimulating the PI3K/AKT signaling pathway. This knowledge deepened our understanding of pancreatic cancer's molecular progression and unveiled potential therapeutic strategies targeting SPDEF-driven pathways.

Akkermansia muciniphila Is Beneficial to a Mouse Model of Parkinson's Disease, via Alleviated Neuroinflammation and Promoted Neurogenesis, with Involvement of SCFAs.

Increasing evidence suggests that the gut microbiota may represent potential strategies for Parkinson's disease (PD) treatment. Our previous research revealed a decreased abundance of Akkermansia muciniphila (Akk) in PD mice; however, whether Akk is beneficial to PD is unknown. To answer this question, the mice received MPTP intraperitoneally to construct a subacute model of PD and were then supplemented with Akk orally for 21 consecutive days. Motor function, dopaminergic neurons, neuroinflammation, and neurogenesis were examined. In addition, intestinal inflammation, and serum and fecal short-chain fatty acids (SCFAs) analyses, were assessed. We found that Akk treatment effectively inhibited the reduction of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and partially improved the motor function in PD mice. Additionally, Akk markedly alleviated neuroinflammation in the striatum and hippocampus and promoted hippocampal neurogenesis. It also decreased the level of colon inflammation. Furthermore, these aforementioned changes are mainly accompanied by alterations in serum and fecal isovaleric acid levels, and lower intestinal permeability. Our research strongly suggests that Akk is a potential neuroprotective agent for PD therapy.

High-throughput extraction and automatic purification of alternariol from edible and medicinal herbs based on aptamer-functionalized magnetic nanoparticles.

Mycotoxin contamination is widespread in plants and herbs, posing serious threats to the consumer and human health. Of them, alternariol (AOH) has attracted great attention as an "emerging" mycotoxin in medicinal herbs. However, a specific and high-throughput extraction method for AOH is currently lacking. Thus, developing an efficient pre-treatment technique for AOH detection is extremely vital. Here, a novel automated magnetic solid-phase extraction method was proposed for the highly efficient extraction of AOH. Combining the aptamer-functionalized magnetic nanoparticles (AMNPs) and the automatic purification instrument, AOH could be extracted in medicinal herbs in high throughput (20 samples) and a short time (30 min). The main parameters affecting extraction were optimized, and the method was finally carried out by incubation AMNPs with 3 mL of sample solution for 10 min, and then desorption in 75% methanol for liquid-phase detection. Under optimal conditions, good reproducibility, stability, and selectivity were realized with an adsorption capacity of 550.84 ng/mg. AOH extraction in three edible herbs showed good resistance to matrix interference with recovery rates from 86% to 111%. In combination with AMNPs and the automatic purification instrument, high-throughput and labor-free extraction of AOH in different complex matrices was achieved, which could be extended in other complex matrices.

Erratum: Author correction to 'Discovery and identification of EIF2AK2 as a direct key target of berberine for anti-inflammatory effects' [Acta Pharmaceutica Sinica B 13 (2023) 2138-2151].

[This corrects the article DOI: 10.1016/j.apsb.2022.12.009.].

A Comprehensive Analysis of Transcriptomics and Metabolomics Revealed Key Pathways Involved in Saccharum spontaneum Defense against Sporisorium scitamineum.

Sugarcane smut, caused by Sporisorium scitamineum, poses a severe threat to sugarcane production. The genetic basis of sugarcane resistance to S. scitamineum remains elusive. A comparative transcriptomic and metabolomic study was conducted on two wild Saccharum species of S. spontaneum with contrast smut resistance. Following infection, the resistant line exhibited greater down-regulation of genes and metabolites compared to the susceptible line, indicating distinct biological processes. Lignan and lignin biosynthesis and SA signal transduction were activated in the resistant line, while flavonoid biosynthesis and auxin signal transduction were enhanced in the susceptible line. TGA2.2 and ARF14 were identified as playing positive and negative roles, respectively, in plant defense. Exogenous auxin application significantly increased the susceptibility of S. spontaneum to S. scitaminum. This study established the significant switching of defense signaling pathways in contrast-resistant S. spontaneum following S. scitamineum infection, offering a hypothetical model and candidate genes for further research into sugarcane smut disease.

Elemental iron protects gut microbiota against oxygen-induced dysbiosis.

Gut dysbiosis induced by oxygen and reactive oxygen species may be related to the development of inflammation, resulting in metabolic syndrome and associated-conditions in the gut. Here we show that elemental iron can serve as an antioxidant and reverse the oxygen-induced dysbiosis. Fecal samples from three healthy donors were fermented with elemental iron and/or oxygen. 16S rRNA analysis revealed that elemental iron reversed the oxygen-induced disruption of Shannon index diversity of the gut microbiota.The bacteria lacking enzymatic antioxidant systems also increased after iron treatment. Inter-individual differences, which corresponded to iron oxidation patterns, were observed for the tested donors. Gut bacteria responding to oxygen and iron treatments were identified as guilds, among which, Escherichia-Shigella was promoted by oxygen and depressed by elemental iron, while changes in bacteria such as Bifidobacterium, Blautia, Eubacterium, Ruminococcaceae, Flavonifractor, Oscillibacter, and Lachnospiraceae were reversed by elemental iron after oxygen treatment. Short-chain fatty acid production was inhibited by oxygen and this effect was partially reversed by elemental iron. These results suggested that elemental iron can regulate the oxygen/ROS state and protect the gut microbiota from oxidative stress.

Rasch analysis of Morse Fall Scale among the older adults with cognitive impairment in nursing homes.

This is a cross-sectional study to investigate the efficacy of Morse Fall Scale (MFS) in nursing homes for older adults with cognitive impairment. According to Rasch analysis, the person separation index was 0.95 (person reliability 0.48), and the item separation index was 9.23 (item reliability 0.99). Wright map showed all items can be considered appropriately directed to the older adults, but the items mainly located at both ends with the center missing. Each item was accepted with good infit and outfit statistics with positive PTMEA CORR. values from 0.49 to 0.68. Two items could be significant differential item functioning (DIF) between the two groups of different fall experience in the past year (item 1 and item 3). In conclusion, nursing assistant could adopt MFS to evaluate fall risk of older adults with cognitive impairment, but the risk grades are still not precise enough. In the future, MFS should be explored and refined further.

A novel frameshift mutation in SOX10 gene induced Waardenburg syndrome type II.

OBJECTIVE: To explore the molecular etiology of Waardenburg syndrome type II (WS2) in a family from Yunnan province, China. METHODS: A total of 406 genes related to hereditary hearing loss were sequenced using next-generation sequencing. DNA samples were isolated from the peripheral blood DNA of probands. Those pathogenic mutations detected by next-generation sequencing in probands and their parents were validated by Sanger sequencing. The conservatism of variation sites in genes was also analyzed. The protein expression was detected by flow cytometry. RESULTS: A heterozygous mutation c.178delG (p.D60fs*49) in the SOX10 gene was identified in the proband, which is a frameshift mutation and may cause protein loss of function and considered to be a pathogenic mutation. This was determined to be a de novo mutation because her family were demonstrated to be wild-type and symptom free. SOX10, FGFR3, SOX2, and PAX3 protein levels were reduced as determined by flow cytometry. CONCLUSION: A novel frameshift mutation in SOX10 gene was identified in this study, which may be the cause of WS2 in proband. In addition, FGFR3, SOX2, and PAX3 might also participate in promoting the progression of WS2.

New insights about endometriosis-associated ovarian cancer: pathogenesis, risk factors, prediction and diagnosis and treatment.

Previous studies have shown that the risk of malignant transformation of endometriosis in premenopausal women is approximately 1%, significantly impacting the overall well-being and quality of life of affected women. Presently, the diagnostic gold standard for endometriosis-associated ovarian cancer (EAOC) continues to be invasive laparoscopy followed by histological examination. However, the application of this technique is limited due to its high cost, highlighting the importance of identifying a non-invasive diagnostic approach. Therefore, there is a critical need to explore non-invasive diagnostic methods to improve diagnostic precision and optimize clinical outcomes for patients. This review presents a comprehensive survey of the current progress in comprehending the pathogenesis of malignant transformation in endometriosis. Furthermore, it examines the most recent research discoveries concerning the diagnosis of EAOC and emphasizes potential targets for therapeutic intervention. The ultimate objective is to improve prevention, early detection, precise diagnosis, and treatment approaches, thereby optimizing the clinical outcomes for patients.

Carbon dot-based fluorescent probe for early diagnosis of pheochromocytoma through identification of circulating tumor cells.

Pheochromocytoma (PCC), as a rare neuroendocrine tumor, is often missed or misdiagnosed because of its atypical clinical manifestations. To realize the early accurate diagnosis of PCC, we have selected circulating tumor cells (CTCs) with more complete biological information as biomarkers and developed a simple and novel fluorescence cytosensor. Octreotide-2,2',2'',2'''- (1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) modified magnetic Fe3O4 and signal amplification CDs@SiO2 nanospheres are prepared to capture and detect PCC-CTCs from peripheral blood via binding to the somatostatin receptor SSTR2 overexpressed on the surface of PCC cells. During the detection process, the target cells were separated and enriched by magnetic capture probes (Fe3O4-DOTA), and then signal probes (CDs@SiO2-DOTA) could also specifically bound to target cells to form the sandwich-like structure for fluorescence signal output. The proposed fluorescence cytosensor has revealed good sensitivity and selectivity for quantitative analysis of PCC-CTCs in the concentration of 5-1000 cells mL-1 with a LOD of 2 cells mL-1. More importantly, designed fluorescence cytosensor has shown good reliability and stability in complex serum samples. This strategy provides a new way for detection of PCC-CTCs.

Inhibition of the HMGB1/RAGE axis protects against cisplatin-induced ototoxicity via suppression of inflammation and oxidative stress.

As an anti-tumor drug widely used in the clinic, cisplatin is limited by its ototoxic side effects associated with various factors, including inflammatory responses. Receptor for Advanced Glycation Endproducts (RAGE) recognizes damage-associated molecular patterns (DAMPs) and promotes stress and inflammation. This study intended to determine the potential behavior of the HMGB1/RAGE axis after cisplatin injury and whether it has a protective effect after inhibiting this pathway. We used FPS-ZM1, a RAGE inhibitor, to modulate the axis of HMGB1/RAGE in neonatal mouse cochlear explants and C57BL/6 mice in vivo. Apoptosis was identified by Annexin V-FITC/PI assay, Cleaved Caspase-3, and TUNEL staining. Reactive oxygen species (ROS) level was assessed by MitoSOX Red and CellROX Green assay. The expression of proteins associated with the HMGB1/RAGE axis and apoptosis was observed by western blotting. The expression of inflammatory cytokines was evaluated by qPCR. The protective effect of HMGB1/RAGE knockdown was also assessed on cisplatin-induced ototoxicity. These results demonstrated that cisplatin could activate the HMGB1/RAGE pathway in cochlear hair cells and release inflammatory factors. Pretreatment with FPS-ZM1 alleviated cisplatin-induced ototoxicity in vivo and in vitro. Knocking down HMGB1 and RAGE achieved specific protective effects. Altogether, inhibiting HMGB1/RAGE axis can reverse the increase of ROS accumulation, the activation of apoptosis, and the production of inflammatory reactions after cisplatin injury. FPS-ZM1 could resist the ototoxicity of cisplatin by suppressing the HMGB1/RAGE signal pathway, and it may be considered the new otoprotective potential strategy for hearing loss.

Ridge preservation in periodontally compromised molar sockets with and without primary wound closure: A comparative controlled clinical trial.

OBJECTIVE: This study aimed to compare hard- and soft-tissue changes after ridge preservation in periodontally compromised molar sockets with and without primary wound closure. MATERIALS AND METHODS: Forty molars with severe periodontitis requiring extraction were included and allocated to two treatment modalities. After tooth extraction, the sockets were filled with deproteinized bovine bone mineral and covered with a bioabsorbable porcine collagen membrane. Primary wound closure was achieved in the control group, whereas the test group underwent minimally invasive open healing. The dimensions of the bone and soft tissue were recorded at baseline and 6 months. RESULTS: Over 6 months, the control and test groups had similar mean ridge heights at the center of sockets of 8.59 ± 2.47 mm and 8.47 ± 2.51 mm, respectively. The total volume of the control group increased from 1070.17 to 1713.52 mm3 for a mean gain of 643.35 mm3 , whereas that of the test group increased from 992.51 to 1514.05 mm3 for a mean gain of 521.54 mm3 . Compared with the test group, the control group showed a statistically significant decrease in keratinized tissue width of 1.08 ± 1.63 mm. CONCLUSIONS: Bone dimensional changes following ridge preservation with and without primary wound closure were comparable. ARP without primary wound closure preserves more keratinized tissue than that with (Chinese Clinical Trial Registry: ChiCTR-ONN-16009433).

sPDGFRß and neuroinflammation are associated with AD biomarkers and differ by race: The ASCEND Study.

INTRODUCTION: There remains an urgent need to identify preclinical pathophysiological mechanisms of Alzheimer's disease (AD) development in high-risk, racially diverse populations. We explored the relationship between cerebrospinal fluid (CSF) markers of vascular injury and neuroinflammation with AD biomarkers in middle-aged Black/African American (B/AA) and non-Hispanic White (NHW) participants. METHODS: Adults (45-65 years) with a parental history of AD were enrolled (n = 82). CSF and blood biomarkers were collected at baseline and year 2. RESULTS: CSF total tau (t-tau), phosphorylated tau (p-tau), and amyloid beta (Aß)40 were elevated at year 2 compared to baseline. CSF soluble platelet-derived growth factor receptor ß (sPDGFRß) levels, a marker of pericyte injury, correlated positively with t-tau, p-tau, Aß40 markers of vascular injury, and cytokines at baseline and year 2. CSF sPDGFRß and tau were significantly lower in B/AA than NHW. DISCUSSION: Vascular dysfunction and neuroinflammation may precede cognitive decline and disease pathology in the very early preclinical stages of AD, and there are race-related differences in these relationships. HIGHLIGHTS: Cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers changed over 2 years in high-risk middle-aged adults. Markers of vascular dysfunction were associated with the CSF biomarkers amyloid beta and tau. AD biomarkers were lower in Black compared to non-Hispanic White individuals. Markers of vascular dysfunction were lower among Black individuals.

Tuberculosis-targeted next-generation sequencing and machine learning: An ultrasensitive diagnostic strategy for paucibacillary pulmonary tuberculosis and tuberculous meningitis.

BACKGROUND: Existing diagnostic approaches for paucibacillary tuberculosis (TB) are limited by the low sensitivity of testing methods and difficulty in obtaining suitable samples. METHODS: An ultrasensitive TB diagnostic strategy was established, integrating efficient and specific TB targeted next-generation sequencing and machine learning models, and validated in clinical cohorts to test plasma cfDNA, cerebrospinal fluid (CSF) DNA collected from tuberculous meningitis (TBM) and pediatric pulmonary TB (PPTB) patients. RESULTS: In the detection of 227 samples, application of the specific thresholds of CSF DNA (AUC = 0.974) and plasma cfDNA (AUC = 0.908) yielded sensitivity of 97.01 % and the specificity of 95.65 % in CSF samples and sensitivity of 82.61 % and specificity of 86.36 % in plasma samples, respectively. In the analysis of 44 paired samples from TBM patients, our strategy had a high concordance of 90.91 % (40/44) in plasma cfDNA and CSF DNA with both sensitivity of 95.45 % (42/44). In the PPTB patient, the sensitivity of the TB diagnostic strategy yielded higher sensitivity on plasma specimen than Xpert assay on gastric lavage (28.57 % VS. 15.38 %). CONCLUSIONS: Our TB diagnostic strategy provides greater detection sensitivity for paucibacillary TB, while plasma cfDNA as an easily collected specimen, could be an appropriate sample type for PTB and TBM diagnosis.