Integrating DNA/RNA microbe detection and host response for accurate diagnosis, treatment and prognosis of childhood infectious meningitis and encephalitis.

BACKGROUND: Infectious meningitis/encephalitis (IM) is a severe neurological disease that can be caused by bacterial, viral, and fungal pathogens. IM suffers high morbidity, mortality, and sequelae in childhood. Metagenomic next-generation sequencing (mNGS) can potentially improve IM outcomes by sequencing both pathogen and host responses and increasing the diagnosis accuracy. METHODS: Here we developed an optimized mNGS pipeline named comprehensive mNGS (c-mNGS) to monitor DNA/RNA pathogens and host responses simultaneously and applied it to 142 cerebrospinal fluid samples. According to retrospective diagnosis, these samples were classified into three categories: confirmed infectious meningitis/encephalitis (CIM), suspected infectious meningitis/encephalitis (SIM), and noninfectious controls (CTRL). RESULTS: Our pipeline outperformed conventional methods and identified RNA viruses such as Echovirus E30 and etiologic pathogens such as HHV-7, which would not be clinically identified via conventional methods. Based on the results of the c-mNGS pipeline, we successfully detected antibiotic resistance genes related to common antibiotics for treating Escherichia coli, Acinetobacter baumannii, and Group B Streptococcus. Further, we identified differentially expressed genes in hosts of bacterial meningitis (BM) and viral meningitis/encephalitis (VM). We used these genes to build a machine-learning model to pinpoint sample contaminations. Similarly, we also built a model to predict poor prognosis in BM. CONCLUSIONS: This study developed an mNGS-based pipeline for IM which measures both DNA/RNA pathogens and host gene expression in a single assay. The pipeline allows detecting more viruses, predicting antibiotic resistance, pinpointing contaminations, and evaluating prognosis. Given the comparable cost to conventional mNGS, our pipeline can become a routine test for IM.

Comparison and development of a metagenomic next-generation sequencing protocol for combined detection of DNA and RNA pathogens in cerebrospinal fluid.

BACKGROUND: The purpose of this study was to evaluate different pretreatment, extraction, amplification, and library generation methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) and to develop an efficient procedure for the simultaneous detection of DNA and RNA pathogens. METHODS: We generated thirteen mock CSF samples with four representative pathogens of encephalitis. Each sample was subjected to ten different methods by varying sample pretreatment/nucleic acid extraction (microbial DNA, total DNA, total NA, total RNA, Whole Transcriptome Amplification (WTA)) and library generation (Illumina or NEB). Negative extraction controls (NECs) were used for each method variation. RESULTS: We found that the quality of mNGS sequencing reads was higher from the NEB kit for library generation. Microbial DNA and total RNA increased microbial deposition by depleting the host DNA. Methods total NA and total RNA can detect gram-positive, gram-negative, RNA and DNA pathogens. We applied mNGS, including total NA and NEB library generation, to CSF samples from five patients diagnosed with infectious encephalitis and correctly determined all pathogens identified in clinical etiological tests. CONCLUSIONS: Our findings suggested that total nucleic acid extraction combined with NEB library generation is the most effective mNGS procedure in CSF pathogen detection. The optimization of positive criteria and databases can improve the specificity and sensitivity of mNGS diagnosis. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800015425 (29/03/2018), https://www.chictr.org.cn/edit.aspx?pid=26292&htm=4 .

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/µL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

Performance of targeted next-generation sequencing in the detection of respiratory pathogens and antimicrobial resistance genes for children.

Introduction. Respiratory tract infection, which is associated with high morbidity and mortality, occurs frequently in children. At present, the main diagnostic method is culture. However, the low pathogen detection rate of the culture approach prevents timely and accurate diagnosis. Fortunately, next-generation sequencing (NGS) can compensate for the deficiency of culture, and its application in clinical diagnostics has become increasingly available.Gap Statement. Targeted NGS (tNGS) is a platform that can select and enrich specific regions before data enter the NGS pipeline. However, the performance of tNGS in the detection of respiratory pathogens and antimicrobial resistance genes (ARGs) in infections in children is unclear.Aim and methodology. In this study, we estimated the performance of tNGS in the detection of respiratory pathogens and ARGs in 47 bronchoalveolar lavage fluid (BALF) specimens from children using conventional culture and antimicrobial susceptibility testing (AST) as the gold standard.Results. RPIP (Respiratory Pathogen ID/AMR enrichment) sequencing generated almost 500 000 reads for each specimen. In the detection of pathogens, RPIP sequencing showed targeted superiority in detecting difficult-to-culture bacteria, including Mycoplasma pneumoniae. Compared with the results of culture, the sensitivity and specificity of RPIP were 84.4 % (confidence interval 70.5-93.5 %) and 97.7 % (95.9 -98.8%), respectively. Moreover, RPIP results showed that a single infection was detected in 10 of the 47 BALF specimens, and multiple infections were detected in 34, with the largest number of bacterial/viral coinfections. Nevertheless, there were also three specimens where no pathogen was detected. Furthermore, we analysed the drug resistance genes of specimens containing Streptococcus pneumoniae, which was detected in 25 out of 47 specimens in the study. A total of 58 ARGs associated with tetracycline, macrolide-lincosamide-streptogramin, beta-lactams, sulfonamide and aminoglycosides were identified by RPIP in 19 of 25 patients. Using the results of AST as a standard, the coincidence rates of erythromycin, tetracycline, penicillin and sulfonamides were 89.5, 79.0, 36.8 and 42.1 %, respectively.Conclusion. These results demonstrated the superiority of RPIP in pathogen detection, particularly for multiple and difficult-to-culture pathogens, as well as in predicting resistance to erythromycin and tetracycline, which has significance for the accurate diagnosis of pathogenic infection and in the guidance of clinical treatment.

Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens.

Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)|-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens.