A multicenter prospective study of comprehensive metagenomic and transcriptomic signatures for predicting outcomes of patients with severe community-acquired pneumonia.

BACKGROUND: Severe community-acquired pneumonia (SCAP) results in high mortality as well as massive economic burden worldwide, yet limited knowledge of the bio-signatures related to prognosis has hindered the improvement of clinical outcomes. Pathogen, microbes and host are three vital elements in inflammations and infections. This study aims to discover the specific and sensitive biomarkers to predict outcomes of SCAP patients. METHODS: In this study, we applied a combined metagenomic and transcriptomic screening approach to clinical specimens gathered from 275 SCAP patients of a multicentre, prospective study. FINDINGS: We found that 30-day mortality might be independent of pathogen category or microbial diversity, while significant difference in host gene expression pattern presented between 30-day mortality group and the survival group. Twelve outcome-related clinical characteristics were identified in our study. The underlying host response was evaluated and enrichment of genes related to cell activation, immune modulation, inflammatory and metabolism were identified. Notably, omics data, clinical features and parameters were integrated to develop a model with six signatures for predicting 30-day mortality, showing an AUC of 0.953 (95% CI: 0.92-0.98). INTERPRETATION: In summary, our study linked clinical characteristics and underlying multi-omics bio-signatures to the differential outcomes of patients with SCAP. The establishment of a comprehensive predictive model will be helpful for future improvement of treatment strategies and prognosis with SCAP. FUNDING: National Natural Science Foundation of China (No. 82161138018), Shanghai Municipal Key Clinical Specialty (shslczdzk02202), Shanghai Top-Priority Clinical Key Disciplines Construction Project (2017ZZ02014), Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100).

The Feasibility of Metagenomic Next-Generation Sequencing to Identify Pathogens Causing Tuberculous Meningitis in Cerebrospinal Fluid.

PURPOSE: The application of metagenomic next-generation sequencing (mNGS) in the diagnosis of tuberculous meningitis (TBM) remains poorly characterized. Here, we retrospectively analyzed data from patients with TBM who had taken both mNGS and conventional tests including culture of Mycobacterium tuberculosis (MTB), polymerase chain reaction (PCR) and acid-fast bacillus (AFB) stain, and the sensitivity and specificity of these methods were compared. METHODS: We retrospectively recruited TBM patients admitted to the hospital between December 2015 and October 2018. The first collection of cerebrospinal fluid (CSF) samples underwent both mNGS and conventional tests. In addition, patients with bacterial/cryptococcal meningitis or viral meningoencephalitis were mNGS positive controls, and a patient with auto-immune encephalitis was an mNGS negative control. RESULTS: Twenty three TBM patients were classified as 12 definite and 11 clinical diagnoses, which were based on clinical manifestations, pathogen evidence, CSF parameters, brain imaging, and treatment response. The mNGS method identified sequences of Mycobacterium tuberculosis complex (MBTC) from 18 samples (18/23, 78.26%). In patients with definite TBM, the sensitivity of mNGS, AFB, PCR, and culture to detect MTB in the first CSF samples were 66.67, 33.33, 25, and 8.33%, respectively. The specificity of each method was 100%. Among the four negative mNGS cases (4/23, 17.39%), three turned out positive by repeated AFB stain. The agreement of mNGS with the total of conventional methods was 44.44% (8/18). Combination of mNGS and conventional methods increased the detection rate to 95.65%. One patient was diagnosed as complex of TBM and cryptococcal meningitis, in which AFB stain and cryptococcal antigen enzyme immunoassay were positive and the DNA of Cryptococcus neoformans was detected by mNGS. CONCLUSION: Our study indicates that mNGS is an alternative method to detect the presence of mycobacterial DNA in CSF samples from patients with TBM and deserves to be applied as a front-line CSF test.

Metagenomic next-generation sequencing contribution in identifying prosthetic joint infection due to Parvimonas micra: a case report.

Identifying fastidious pathogens in patients with prosthetic joint infection (PJI) by culture is challenging. Metagenomic next-generation sequencing (mNGS) is a novel culture-independent approach that is associated with a higher likelihood for identifying pathogens. We present a case where mNGS was implemented to identify Parvimonas micra, a rarely reported and difficult-to-culture PJI pathogen.

Development and validation of a new HPV genotyping assay based on next-generation sequencing.

OBJECTIVES: We developed a new human papillomavirus (HPV) genotyping assay based on multiplex polymerase chain reaction and next-generation sequencing (NGS) methods for large-scale cervical cancer screening. METHODS: We first trained the assay on 1,170 self-collected samples, balancing the cutoff points for high-risk types. Then using 4,262 separate self-collected specimens, we compared concordance, sensitivity, and specificity for cervical intraepithelial neoplasia type 2 (CIN2) or higher and CIN type 3 (CIN3) or higher of the HPV sequencing assay with that of Hybrid Capture 2 (HC2) direct samples and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay self-samples. RESULTS: All assays had a good agreement. The sensitivity for CIN2 or higher and CIN3 or higher of the self-sampling specimens tested with the sequencing assay run on both MiSeq and Ion Torrent Personal Genome Machine sequencer was similar to that of direct-sampling specimens tested with HC2 (P > .05), but the specificity of the sequencing assay for CIN2 or higher and CIN3 or higher was significantly higher than that of HC2 (P < .01). CONCLUSIONS: This population-based study has demonstrated the applicability of a new NGS high-risk HPV assay for primary cervical cancer screening based on self-collection.

A new PCR-based mass spectrometry system for high-risk HPV, part I: methods.

Infection with high-risk (HR) human papillomaviruses (HPVs) has been confirmed as the necessary cause of cervical cancer. There are many studies that have established and confirmed the relationship of specific HPV types and the risk of invasive cervical cancer. We have developed a novel genotyping method for detecting 14 HR-HPV genotypes simultaneously with MassARRAY (Sequenom, San Diego, CA) technique based on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). All 14 HPVs showed high specificities and high sensitivities in the plasmid test; lower detection limits for each genotype were from 10 to 100 copies. Furthermore, the MS system has high-throughput capacities, capable of processing, with type-specific output, 4,500 samples in 24 hours. The MS HPV assay is a sensitive and useful tool for HPV genotyping. It has the potential to be suitable for large-scale epidemiologic studies and routine diagnostic clinical applications owing to its high-throughput capacity, high sensitivity, and low cost per case.

Sequencing of 50 human exomes reveals adaptation to high altitude.

Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP's association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.