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.

UNASSIGNED: Metagenomic next-generation sequencing (mNGS), which provides untargeted and unbiased pathogens detection, has been extensively applied to improve diagnosis of pulmonary infection. This study aimed to compare the clinical performance between mNGS and targeted NGS (tNGS) for microbial detection and identification in bronchoalveolar lavage fluid (BALF) from kidney transplantation recipients (KTRs).
UNASSIGNED: BALF samples with microbiological results from mNGS and conventional microbiological test (CMT) were included. For tNGS, samples were extracted, amplified by polymerase chain reaction with pathogen-specific primers, and sequenced on an Illumina Nextseq.
UNASSIGNED: A total of 99 BALF from 99 KTRs, among which 93 were diagnosed as pulmonary infection, were analyzed. Compared with CMT, both mNGS and tNGS showed higher positive rate and sensitivity (p<0.001) for overall, bacterial and fungal detection. Although the positive rate for mNGS and tNGS was comparable, mNGS significantly outperformed tNGS in sensitivity (100% vs. 93.55%, p<0.05), particularly for bacteria and virus (p<0.001). Moreover, the true positive rate for detected microbes of mNGS was superior over that of tNGS (73.97% vs. 63.15%, p<0.05), and the difference was also significant when specific for bacteria (94.59% vs. 64.81%, p<0.001) and fungi (93.85% vs. 72.58%, p<0.01). Additionally, we found that, unlike most microbes such as SARS-CoV-2, Aspergillus, and EBV, which were predominantly detected from recipients who underwent surgery over 3 years, Torque teno virus (TTV) were principally detected from recipients within 1-year post-transplant, and as post-transplantation time increased, the percentage of TTV positivity declined.
UNASSIGNED: Although tNGS was inferior to mNGS owing to lower sensitivity and true positive rate in identifying respiratory pathogens among KTRs, both considerably outperformed CMT.

BACKGROUND: Plastic bronchitis (PB) is an uncommon and severe acute respiratory ailment characterized by the formation of casts in the trachea or bronchial tree. Some instances have been linked to human bocavirus (HBoV) infections.
METHODS: In this report, we present a case of PB secondary to HBoV1 infection in a previously healthy pediatric patient. A 17-month-old male was admitted due to respiratory distress following 2 days of cough and fever. A preadmission chest X-ray revealed atelectasis of the left lung. Emergency electronic bronchoscopy and foreign body forceps were employed to remove casts, leading to improved breathing. High-throughput next-generation sequencing detected only HBoV1. A subsequent electronic bronchoscopy 2 days later showed no casts.
CONCLUSIONS: PB associated with HBoV1 infection should be considered in children experiencing acute respiratory distress, and a second bronchoscopy intervention may not be necessary in cases related to HBoV1.

Conidiobolus sensu lato, a genus within the family Ancylistaceae, encompasses a diverse range of fungal species that are widely distributed in plant debris and soil. In this study, we identified three double-stranded RNA (dsRNA) viruses coinfecting a strain of Conidiobolus taihushanensis. These viruses were identified as Conidiobolus taihushanensis totivirus 1 (CtTV1), Conidiobolus nonsegmented RNA virus 1-2 (CNRV1-2), and Conidiobolus taihushanensis virus 1 (CtV1). Through high-throughput sequencing and RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we determined their complete genome sequences. The genome of CtTV1 is 6,921 nucleotides in length, containing two open reading frames (ORFs). ORF1 encodes a 1,124-amino-acid capsid protein (CP) with a molecular weight of 125.07 kDa, and ORF2 encodes a 780-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular weight of 88.05 kDa. CNRV1-2, approximately 3.0 kb in length, also contains two ORFs, which are predicted to encode a 186-amino-acid hypothetical protein (HP) and a 758-amino-acid RdRp. CtV1 has a smaller genome consisting of 3,081 base pairs (bp) with two ORFs: one encoding a 244-amino-acid HP (26.85 kDa) and the other encoding a 707-amino-acid RdRp (80.64 kDa). Phylogenetic analysis based on RdRp sequences revealed that CtTV1 shows the highest similarity to Phytophthora pluvialis RNA virus 1, with 38.79% sequence identity, and clusters with members of the family Orthototiviridae, and it is most closely related to Utsjoki toti-like virus. In contrast, CtV1 formed a unique branch and might represent a new genus. The genome sequence of CNRV1-2 is 99.74% identical to that of the previously described Conidiobolus non-segmented RNA virus 1 (CNRV1). Our findings indicate that CtTV1 and CtV1 are distinct novel viruses, while CNRV1-2 appears to be a variant of CNRV1. This study enhances our understanding of the genetic diversity and evolutionary relationships among mycoviruses associated with C. taihushanensis.

BACKGROUND: Newborn screening (NBS) for primary carnitine deficiency (PCD) has poor performance. This study aimed to evaluate the feasibility of incorporating next-generation sequencing (NGS) as a second-tier PCD test.
METHODS: Between March and December 2020, 60,070 newborns were screened for inherited metabolic disorders. Newborns with free carnitine (C0) levels below 8.5 μmol/L were selected for second-tier genetic testing.
RESULTS: In total, 130 (0.22%) newborns with low C0 levels underwent second-tier genetic testing, 87 (66.92%) had positive genetic testing results, and 30 (23.08%) carried pathogenic variants of the SLC22A5 gene. Six newborns were diagnosed with PCD. The incidence of PCD was approximately 1 in 1:10,012 newborns. The PPV reached 20% after combining with second-tier NGS. Of the eight variants identified in patients with PCD, the three most common variants were c.760C>T (p.Arg254*), c.51C>G (p.Phe17Leu), and c.1400C>G (p.Ser467Cys). The C0 levels of patients with PCD were significantly lower than those of PCD carriers (p = 0.0026) and PCD-negative individuals (p = 0.0005).
CONCLUSIONS: Our results showed that the PPV reached 20% after combining with second-tier NGS. The MS/MS-based NBS and second-tier NGS combination can effectively reduce the false-positive rate and detect PCD in patients.

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.

In forensic genetics, utilizing massively parallel sequencing (MPS) to analyze short tandem repeats (STRs) has demonstrated several advantages compared to conventional capillary electrophoresis (CE). Due to the current technical limitations, although flanking region polymorphisms had been mentioned in several previous studies, most studies focused on the core repeat regions of STRs or the variations in the adjacent flanking regions. In this study, we developed an MPS system consisting of two sets of multiplex PCR systems to detect not only the STR core repeat regions but also to observe variants located at relatively distant positions in the flanking regions. The system contained 42 commonly used forensic STRs, including 21 autosomal STRs (A-STRs) and 21 Y-chromosomal STRs (Y-STRs), and a total of 350 male individuals from a Chinese Han population were genotyped. The length and sequence variants per locus were tallied and categorized based on length (length-based, LB), sequence without flanking region (core repeat regions sequence-based, RSB), and sequence with flanking region (core repeat and flanking regions sequence-based, FSB), respectively. Allele frequencies, Y-haplotype frequencies, and forensic parameters were calculated based on LB, RSB, and FSB, respectively, to evaluate the improvement in discrimination power, heterozygosity, and effectiveness of forensic systems. The results suggested the sequence variations have more influence on A-STRs and could improve the identification ability of MPS-STR genotyping. Concordance between MPS and CE methods was confirmed by using commercial CE-based STR kits. The impact of flanking region variations on STR genotype analysis and potential factors contributing to discordances were discussed. A total of 58 variations in the flanking regions (53 SNPs/SNVs and 5 InDels) were observed and most variations (48/58) were distributed in A-STRs. In summary, this study delved deeper into the genetic information of forensic commonly used STR and advanced the application of massively parallel sequencing in forensic genetics.

BACKGROUND: T cells, the \"superstar\" of the immune system, play a crucial role in antitumor immunity. T-cell receptors (TCR) are crucial molecules that enable T cells to identify antigens and start immunological responses. The body has evolved a unique method for rearrangement, resulting in a vast diversity of TCR repertoires. A healthy TCR repertoire is essential for the particular identification of antigens by T cells.
METHODS: In this article, we systematically summarized the TCR creation mechanisms and analysis methodologies, particularly focusing on the application of next-generation sequencing (NGS) technology. We explore the TCR repertoire in health and cancer, and discuss the implications of TCR repertoire analysis in understanding carcinogenesis, cancer progression, and treatment.
RESULTS: The TCR repertoire analysis has enormous potential for monitoring the emergence and progression of malignancies, as well as assessing therapy response and prognosis. The application of NGS has dramatically accelerated our comprehension of TCR diversity and its role in cancer immunity.
CONCLUSIONS: To substantiate the significance of TCR repertoires as biomarkers, more thorough and exhaustive research should be conducted. The TCR repertoire analysis, enabled by advanced sequencing technologies, is poised to become a crucial tool in the future of cancer diagnosis, monitoring, and therapy evaluation.

BACKGROUND: Rapid proliferation and outgrowth of tumor cells frequently result in localized hypoxia, which has been implicated in the progression of lung cancer. The present study aimed to identify key long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in hypoxia-induced A549 lung cancer cells, and to investigate their potential underlying mechanisms of action.
METHODS: High-throughput sequencing was utilized to obtain the expression profiles of lncRNA and mRNA in both hypoxia-induced and normoxia A549 lung cancer cells. Subsequently, a bioinformatics analysis was conducted on the differentially expressed molecules, encompassing functional enrichment analysis, protein-protein interaction (PPI) network analysis, and competitive endogenous RNA (ceRNA) analysis. Finally, the alterations in the expression of key lncRNAs and mRNAs were validated using real-time quantitative PCR (qPCR).
RESULTS: In the study, 1155 mRNAs and 215 lncRNAs were identified as differentially expressed between the hypoxia group and the normoxia group. Functional enrichment analysis revealed that the differentially expressed mRNAs were significantly enriched in various pathways, including the p53 signaling pathway, DNA replication, and the cell cycle. Additionally, key lncRNA-miRNA-mRNA relationships, such as RP11-58O9.2-hsa-miR-6749-3p-XRCC2 and SNAP25-AS1-hsa-miR-6749-3p-TENM4, were identified. Notably, the qPCR assay demonstrated that the expression of SNAP25-AS1, RP11-58O9.2, TENM4, and XRCC2 was downregulated in the hypoxia group compared to the normoxia group. Conversely, the expression of LINC01164, VLDLR-AS1, RP11-14I17.2, and CDKN1A was upregulated.
CONCLUSIONS: Our findings suggest a potential involvement of SNAP25-AS1, RP11-58O9.2, TENM4, XRCC2, LINC01164, VLDLR-AS1, RP11-14I17.2, and CDKN1A in the development of hypoxia-induced lung cancer. These key lncRNAs and mRNAs exert their functions through diverse mechanisms, including the competitive endogenous RNA (ceRNA) pathway.

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.