UNASSIGNED: To investigate the diagnostic value of metagenomic next-generation sequencing (mNGS) in detecting pathogens from joint infection (JI) synovial fluid (SF) samples with previous antibiotic exposure.
UNASSIGNED: From January 2019 to January 2022, 59 cases with suspected JI were enrolled. All cases had antibiotic exposure within 2 weeks before sample collection. mNGS and conventional culture were performed on SF samples. JI was diagnosed based on history and clinical symptoms in conjunction with MSIS criteria. The diagnostic values, including sensitivity, specificity, positive/negative predictive values (PPV/NPV), and accuracy, were in comparison with mNGS and culture.
UNASSIGNED: There were 47 of the 59 cases diagnosed with JI, while the remaining 12 were diagnosed with non-infectious diseases. The sensitivity of mNGS was 68.1%, which was significantly higher than that of culture (25.5%, p<0.01). The accuracy of mNGS was significantly higher at 71.2% compared to the culture at 39.0% (p <0.01). Eleven pathogenic strains were detected by mNGS but not by microbiological culture, which included Staphylococcus lugdunensis, Staphylococcus cohnii, Finegoldia magna, Enterococcus faecalis, Staphylococcus saprophytics, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter pittii, Brucella ovis, andCoxiella burnetii. Antibiotic therapy was adjusted based on the mNGS results in 32 (68.1%) patients, including 12 (25.5%) and 20 (42.6%) patients, in whom treatment was upgraded and changed, respectively. All JI patients underwent surgery and received subsequent antibiotic therapy. They were followed up for an average of 23 months (20-27 months), and the success rate of treatment was 89.4%. Out of the 33 patients who had positive results for pathogens, reoperation was performed in 1 case (3.03%), while out of the 14 cases with negative results for both mNGS and cultures, reoperation was performed in 4 cases (28.6%).
UNASSIGNED: mNGS has advantages over conventional culture in detecting pathogens in SF samples from JI patients previously treated with antibiotics, potentially improving clinical outcomes.

BACKGROUND: Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity.
METHODS: We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers.
RESULTS: Capture with the Twist CVRP increased sensitivity by at least 10-100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600-6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT.
CONCLUSIONS: Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.

BACKGROUND: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.
RESULTS: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).
CONCLUSIONS: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. Video Abstract.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is diagnosed relatively late and has a poor prognosis, requiring early detection to reduce the disease burden. This diagnostic test accuracy meta-analysis evaluated the serological diagnostic value of nine EBV-related IgA antibody panels (EBNA1-IgA, VCA-IgA, EA-IgA, Zta-IgA, EBNA1-IgA + VCA-IgA, VCA-IgA + EA-IgA, VCA-IgA + Rta-IgG, EBNA1-IgA + VCA-IgA + Zta-IgA and VCA-IgA + EA-IgA + Rta-IgG), aiming to identify suitable serological detection biomarkers for NPC screening.
METHODS: PubMed, Embase, China National Knowledge Infrastructure and Chinese BioMedical Literature Database were searched from January 1st, 2000 to September 30th, 2023, with keywords nasopharyngeal carcinoma, IgA, screening, early detection, early diagnosis, sensitivity and specificity. Articles on the diagnostic value of serum EBV-related IgA antibody panels for NPC were included. Study selection, data extraction, and quality assessment were performed independently by two researchers, and a third researcher was consulted in the case of disagreement. Bivariate models were used for statistical analysis. The quality of included studies was evaluated through Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2).
RESULTS: A total of 70 articles were included, involving 11 863 NPC cases and 34 995 controls. Among the nine EBV-related IgA antibody panels, EBNA1-IgA + VCA-IgA [0.928 (0.898, 0.950)], VCA-IgA + Rta-IgG [0.925 (0.890, 0.949)], EBNA1-IgA + VCA-IgA + Zta-IgA [0.962 (0.909, 0.985)] and VCA-IgA + EA-IgA + Rta-IgG [0.945 (0.918, 0.964)] demonstrated higher pooled sensitivity (95%CI). In terms of diagnostic odds ratio (DOR) (95%CI), EBNA1-IgA + VCA-IgA [107.647 (61.173, 189.430)], VCA-IgA + Rta-IgG [105.988 (60.118, 186.857)] and EBNA1-IgA + VCA-IgA + Zta-IgA [344.450 (136.351, 870.153)] showed superior performance. Additionally, the SROC curves for EBNA1-IgA + VCA-IgA and VCA-IgA + Rta-IgG were more favorable. However, publication bias was detected for VCA-IgA (P = 0.005) and EBNA1-IgA + VCA-IgA (P = 0.042).
CONCLUSIONS: In general, parallel detection of serum EBNA1-IgA, VCA-IgA and Zta-IgA antibodies using ELISA demonstrates better pooled sensitivity and DOR among the studied panels. In the cases where fewer indicators are used, serum VCA-IgA and EBNA1-IgA/Rta-IgG antibody panel exhibits a comparable performance.
BACKGROUND: The International Prospective Register of Systematic Reviews registration number: CRD42023426984, registered on May 28, 2023.

OBJECTIVE: Develop a practical scoring system based on radiomics and imaging features, for predicting the malignant potential of incidental indeterminate small solid pulmonary nodules (IISSPNs) smaller than 20 mm.
METHODS: A total of 360 patients with malignant IISSPNs (n = 213) and benign IISSPNs (n = 147) confirmed after surgery were retrospectively analyzed. The whole cohort was randomly divided into training and validation groups at a ratio of 7:3. The least absolute shrinkage and selection operator (LASSO) algorithm was used to debase the dimensions of radiomics features. Multivariate logistic analysis was performed to establish models. The receiver operating characteristic (ROC) curve, area under the curve (AUC), 95% confidence interval (CI), sensitivity and specificity of each model were recorded. Scoring system based on odds ratio was developed.
RESULTS: Three radiomics features were selected for further model establishment. After multivariate logistic analysis, the combined model including Mean, age, emphysema, lobulated and size, reached highest AUC of 0.877 (95%CI: 0.830-0.915), accuracy rate of 83.3%, sensitivity of 85.3% and specificity of 80.2% in the training group, followed by radiomics model (AUC: 0.804) and imaging model (AUC: 0.773). A scoring system with a cutoff value greater than 4 points was developed. If the score was larger than 8 points, the possibility of diagnosing malignant IISSPNs could reach at least 92.7%.
CONCLUSIONS: The combined model demonstrated good diagnostic performance in predicting the malignant potential of IISSPNs. A perfect accuracy rate of 100% can be achieved with a score exceeding 12 points in the user-friendly scoring system.

BACKGROUND: Perioperative reflux aspiration presents a grave concern during sedation or general anesthesia, particularly when solid gastric contents prompt acute upper respiratory obstruction, potentially resulting in fatal consequences for patients. Currently, there are limited means for promptly assessing solid gastric contents in clinical settings. Therefore, this study examined the efficacy of ultrasound assessment for solid gastric contents, offering a rapid and non-invasive approach for early detection and decision-making regarding interventions.
METHODS: The study included 400 patients scheduled for upper endoscopy procedures, which encompassed both gastroscope and gastroscope combined colonoscopy examinations with sedation. Ultrasound scanning of the antrum was performed while patients were positioned semi-sitting or in the right lateral decubitus (RLD) posture. The evaluation of solid gastric contents relied on direct visual observation during endoscopy. Gastric volume measurement occurred subsequent to endoscopic suction of gastric contents. Receiver operating characteristic curves were utilized to assess the effectiveness of ultrasonography in discerning solid contents.
RESULTS: Seven patients undergoing gastroscope with sedation were found to have solid gastric contents. The sensitivity, specificity, positive predictive value, and negative predictive value of the ultrasound qualitative evaluation of solid contents were 85.7%, 99%, 60%, and 99.7%, respectively.
CONCLUSIONS: Solid stomach contents can be evaluated qualitatively with reasonable accuracy using ultrasonography. Additionally, in patients undergoing upper endoscopy and assessed to have solid gastric contents with ultrasound, administration of mild sedation is recommended.
BACKGROUND: www.chictr.org.cn (ChiCTR2100048994); registered 19/07/2021.

BACKGROUND: Malaria control depends primarily on rapid and accurate diagnosis followed by successful treatment. Light microscopy is still used as a gold standard method for the diagnosis of malaria. The Sysmex hematology analyzer is a novel method for malaria detection. Therefore, the aim of this review was to investigate the diagnostic accuracy of the Sysmex hematology analyzer for malaria diagnosis.
METHODS: Electronic databases like PubMed, PubMed Central, Science Direct databases, Google Scholar, and Scopus were used to find relevant articles from April to June 14, 2023. The studies\' methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Using Review Manager 5.4.1, the estimates of sensitivity and specificity, as well as their 95% confidence intervals, were shown in forest plots. Midas software in Stata 14.0 was utilized to calculate the summary estimates of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio. Heterogeneity was assessed by using I2 statistics. In addition, publication bias was assessed using a funnel plot and Deeks\' test. Sub-group and meta- regression analysis were also performed.
RESULTS: A total of 15 studies were assessed for diagnostic accuracy. The sensitivity and specificity of Sysmex hematology analyzer for studies ranged from 46% to 100% and 81% to 100%, respectively. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer were 95% (95% CI: 85%-99%) and 99% (95% CI: 97%-100%), respectively. It had excellent diagnostic accuracy. There were significant heterogeneity among the studies included in this meta-analysis. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer using polymerase chain reaction as the gold standard was 97.6% (95% CI: 83.2, 99.7) and 99.4% (98.5, 99.8), respectively.
CONCLUSIONS: In this review, Sysmex hematology analyzer had excellent diagnostic accuracy. Therefore, it could be used as an alternate diagnostic tool for malaria diagnosis in the hospital and health center.
BACKGROUND: Systematic review registration PROSPERO (2023: CRD42023427713). https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023427713.

Tuberculosis (TB) is the leading cause of mortality among infectious diseases globally. Effectively managing TB requires early identification of individuals with TB disease. Resource-constrained settings often lack skilled professionals for interpreting chest X-rays (CXRs) used in TB diagnosis. To address this challenge, we developed \"DecXpert\" a novel Computer-Aided Detection (CAD) software solution based on deep neural networks for early TB diagnosis from CXRs, aiming to detect subtle abnormalities that may be overlooked by human interpretation alone. This study was conducted on the largest cohort size to date, where the performance of a CAD software (DecXpert version 1.4) was validated against the gold standard molecular diagnostic technique, GeneXpert MTB/RIF, analyzing data from 4363 individuals across 12 primary health care centers and one tertiary hospital in North India. DecXpert demonstrated 88% sensitivity (95% CI 0.85-0.93) and 85% specificity (95% CI 0.82-0.91) for active TB detection. Incorporating demographics, DecXpert achieved an area under the curve of 0.91 (95% CI 0.88-0.94), indicating robust diagnostic performance. Our findings establish DecXpert\'s potential as an accurate, efficient AI solution for early identification of active TB cases. Deployed as a screening tool in resource-limited settings, DecXpert could enable early identification of individuals with TB disease and facilitate effective TB management where skilled radiological interpretation is limited.

OBJECTIVE: Despite global research on early detection of age-related macular degeneration (AMD), not enough is being done for large-scale screening. Automated analysis of retinal images captured via smartphone presents a potential solution; however, to our knowledge, such an artificial intelligence (AI) system has not been evaluated. The study aimed to assess the performance of an AI algorithm in detecting referable AMD on images captured on a portable fundus camera.
METHODS: A retrospective image database from the Age-Related Eye Disease Study (AREDS) and target device was used.
METHODS: The algorithm was trained on two distinct data sets with macula-centric images: initially on 108,251 images (55% referable AMD) from AREDS and then fine-tuned on 1108 images (33% referable AMD) captured on Asian eyes using the target device. The model was designed to indicate the presence of referable AMD (intermediate and advanced AMD). Following the first training step, the test set consisted of 909 images (49% referable AMD). For the fine-tuning step, the test set consisted of 238 (34% referable AMD) images. The reference standard for the AREDS data set was fundus image grading by the central reading centre, and for the target device, it was consensus image grading by specialists.
METHODS: Area under receiver operating curve (AUC), sensitivity and specificity of algorithm.
RESULTS: Before fine-tuning, the deep learning (DL) algorithm exhibited a test set (from AREDS) sensitivity of 93.48% (95% CI: 90.8% to 95.6%), specificity of 82.33% (95% CI: 78.6% to 85.7%) and AUC of 0.965 (95% CI:0.95 to 0.98). After fine-tuning, the DL algorithm displayed a test set (from the target device) sensitivity of 91.25% (95% CI: 82.8% to 96.4%), specificity of 84.18% (95% CI: 77.5% to 89.5%) and AUC 0.947 (95% CI: 0.911 to 0.982).
CONCLUSIONS: The DL algorithm shows promising results in detecting referable AMD from a portable smartphone-based imaging system. This approach can potentially bring effective and affordable AMD screening to underserved areas.

UNASSIGNED: Patients who were infected by the Human Immunodeficiency Virus (HIV) could have weakened immunity that is complicated by opportunistic infections, especially for Mycobacterium tuberculosis (MTB). Notably, the HIV-MTB co-infection will accelerate the course of disease progress and greatly increase the mortality of patients. Since the traditional diagnostic methods are time-consuming and have low sensitivity, we aim to investigate the performance of mNGS (metagenomic Next-Generation Sequencing) and mNPS (metagenomic NanoPore Sequencing) for the rapid diagnosis of tuberculosis in HIV-infected patients.
UNASSIGNED: The 122 HIV-infected patients were enrolled for the retrospective analysis. All of the patients underwent traditional microbiological tests, mNGS, and (or) mNPS tests. The clinical comprehensive diagnosis was used as the reference standard to compare the diagnostic performance of culture, mNGS, and mNPS on tuberculosis. We also investigate the diagnostic value of mNGS and mNPS on mixed-infection. Furthermore, the treatment adjustment directed by mNGS and mNPS was analyzed.
UNASSIGNED: Compared with the composite reference standard, the culture showed 42.6% clinical sensitivity and 100% specificity, and the OMT(other microbiological testing) had 38.9% sensitivity and 100% specificity. The mNGS had 58.6% clinical sensitivity and 96.8% specificity, and the mNPS had 68.0% clinical sensitivity and 100% specificity. The proportion of mixed-infection cases (88.9%) in the TB group was higher than those in the non-TB group (54.8%) and the mNGS and mNPS are more competitive on mixed-infection diagnosis compared with the traditional methods. Furthermore, there are 63 patients (69.2%) and 36 patients (63.2%) achieved effective treatment after receiving the detection of mNPS and mNGS, respectively.
UNASSIGNED: Our study indicated that mNPS and mNGS have high sensitivity and specificity for TB diagnosis compared with the traditional methods, and mNPS seems to have better diagnostic performance than mNGS. Moreover, mNGS and mNPS showed apparent advantages in detecting mixed infection. The mNPS and mNGS-directed medication adjustment have effective treatment outcomes for HIV-infected patients who have lower immunity.