RESUMEN
Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).
Asunto(s)
Bacterias/genética , COVID-19/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus ARN/genética , Infecciones del Sistema Respiratorio/diagnóstico , SARS-CoV-2/genética , Bacterias/aislamiento & purificación , COVID-19/virología , Coronavirus/genética , Coronavirus/aislamiento & purificación , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Nanoporos , Orthomyxoviridae/genética , Orthomyxoviridae/aislamiento & purificación , Virus ARN/aislamiento & purificación , ARN Viral/química , ARN Viral/metabolismo , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , SARS-CoV-2/aislamiento & purificaciónRESUMEN
BACKGROUND: Clinical metagenomics (CMg) has the potential to be translated from a research tool into routine service to improve antimicrobial treatment and infection control decisions. The SARS-CoV-2 pandemic provides added impetus to realise these benefits, given the increased risk of secondary infection and nosocomial transmission of multi-drug-resistant (MDR) pathogens linked with the expansion of critical care capacity. METHODS: CMg using nanopore sequencing was evaluated in a proof-of-concept study on 43 respiratory samples from 34 intubated patients across seven intensive care units (ICUs) over a 9-week period during the first COVID-19 pandemic wave. RESULTS: An 8-h CMg workflow was 92% sensitive (95% CI, 75-99%) and 82% specific (95% CI, 57-96%) for bacterial identification based on culture-positive and culture-negative samples, respectively. CMg sequencing reported the presence or absence of ß-lactam-resistant genes carried by Enterobacterales that would modify the initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus from 4 positive and 39 negative samples. Molecular typing using 24-h sequencing data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak involving 14 patients across three ICUs. CONCLUSION: CMg testing provides accurate pathogen detection and antibiotic resistance prediction in a same-day laboratory workflow, with assembled genomes available the next day for genomic surveillance. The provision of this technology in a service setting could fundamentally change the multi-disciplinary team approach to managing ICU infections. The potential to improve the initial targeted treatment and rapidly detect unsuspected outbreaks of MDR-pathogens justifies further expedited clinical assessment of CMg.