A dual-process of targeted and unbiased Nanopore sequencing enables accurate and rapid diagnosis of lower respiratory infections.

BACKGROUND: Nanopore metagenomics has been used for infectious disease diagnosis for bacterial pathogens. However, this technology currently lacks comprehensive performance studies in clinical settings for simultaneous detection of bacteria, fungi, and viruses. METHODS: We developed a dual-process of Nanopore sequencing for one sample, with unbiased metagenomics in Meta process and target enrichment in Panel process (Nanopore Meta-Panel process, NanoMP) and prospectively enrolled 450 respiratory specimens from multiple centers. The filter system of pathogen detection was established with machine learning and receiver operator characteristic (ROC) curve to optimize the detection accuracy based on orthogonal test of 21 species. Antimicrobial resistance (AMR) genes were identified based on the Comprehensive Antibiotic Resistance Database (CARD) and single-nucleotide polymorphism matrix. FINDINGS: Our approach showed high sensitivity in Meta process, with 82.9%, 88.7%, and 75.0% for bacteria, fungi (except Aspergillus), and Mycobacterium tuberculosis groups, respectively. Moreover, target amplification improved the sensitivity of virus (>80.0% vs. 39.4%) and Aspergillus (81.8% vs. 42.3%) groups in Panel process compared with Meta process. Overall, NanoMP achieved 80.2% sensitivity and 98.8% specificity compared with the composite reference standard, and we were able to accurately detect AMR genes including blaKPC-2, blaOXA-23 and mecA and distinguish their parent organisms in patients with mixed infections. INTERPRETATION: We combined metagenomic and enriched Nanopore sequencing for one sample in parallel. Our NanoMP approach simultaneously covered bacteria, viruses and fungi in respiratory specimens and demonstrated good diagnostic performance in real clinical settings. FUNDING: National Key Research and Development Program of China and National Natural Science Foundation of China.

Molecular Epidemiology and Antifungal Resistance of Cryptococcus neoformans From Human Immunodeficiency Virus-Negative and Human Immunodeficiency Virus-Positive Patients in Eastern China.

Cryptococcosis is an opportunistic and potentially lethal infection caused by Cryptococcus neoformans and Cryptococcus gattii complex, which affects both immunocompromised and immunocompetent people, and it has become a major public health concern worldwide. In this study, we characterized the molecular epidemiology and antifungal susceptibility of 133 C. neoformans isolates from East China Invasive Fungal Infection Group (ECIFIG), 2017-2020. Isolates were identified to species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry and confirmed by IGS1 sequencing. Whole-genome sequencing (WGS) was conducted on three multidrug-resistant isolates. Among the 133 strains, 61 (45.86%) were isolated from HIV-positive patients and 72 (54.16%) were isolated from HIV-negative patients. In total, C. neoformans var. grubii accounted for 97.74% (130/133), while C. neoformans var. neoformans was rare (2.06%, 3/133). The strains were further classified into nine sequence types (STs) dominated by ST5 (90.23%, 120/133) with low genetic diversity. No association was observed between STs and HIV status. All strains were wild type to voriconazole, while high antifungal minimal inhibitory concentrations (MICs) above the epidemiological cutoff values (ECVs) were observed in C. neoformans strains, and more than half of isolates were non-wild-type to amphotericin B (89.15%, 109/133). Eight isolates were resistant to fluconazole, and eight isolates were non-wild type to 5-fluorocytosine. Furthermore, WGS has verified the novel mutations of FUR1 in 5-fluorocytosine-resistant strains. In one isolate, aneuploidy of chromosome 1 with G484S mutation of ERG11 was observed, inducing high-level resistance (MIC: 32 µg/ml) to fluconazole. In general, our data showed that there was no significant difference between HIV-positive and HIV-negative patients on STs, and we elucidate the resistant mechanisms of C. neoformans from different perspectives. It is important for clinical therapy and drug usage in the future.