Abstract
INTRODUCTION: With the emergence and re-emergence of infectious diseases, the need for rapid and accurate detection of the causative pathogen is crucial to provide appropriate therapy. Current gold standard of culture-based detection suffers longer turnaround time (3-5 days), low sensitivity and failure to detect fastidious and rare pathogens(1). Next generation sequencing based diagnosis provides the scope of unbiased and data driven comprehensive detection on a single platform with accuracy, and shorter turnaround time. Routine implementation in the clinical setting will markedly improve real-time point-of-care pathogen diagnosis(2), thus, aiding initiation of optimal therapy, better patient outcomes and shorter hospital stay. OBJECTIVES: Retrospective analysis of pathogens and ARGs to evaluate the relevance of genome sequencing technique as a routine diagnostic test for detection of infections directly from samples of patients in critical care units. MATERIALS AND METHODS: Clinical samples were aseptically collected from patients with sepsis or critical infection of sterile sites. These samples were directly used for genome sequencing assay. Amplicon-based real-time sequencing with targets to identify bacteria, fungus and antimicrobial genes(ARGs) was performed on the Oxford Nanopore sequencing platform. Analysis was performed on a proprietary automated genome sequencing software that generated clinical reports. A retrospective analysis was performed on the pathogens and ARGs identified through the routine implementation of the test. RESULTS: A total of 31 clinical samples were included in the analysis which consisted of 23 plasma, 4 bronchoalveolar lavage, 3 cerebrospinal fluid and 1 pleural fluid. A total of 38 pathogens were detected using sequencing of which 60.5% were gram-negative bacteria, 26.3% were gram-positive bacteria and 13% were fungus. Acinetobacter spp(21.05%) and Escherichia coli (7.89%) were the most frequently detected gram-negative bacteria whereas Micrococcus spp(7.89%) and Kocuria subflava(5.26%) were the most frequent gram-positive bacteria detected. Malassezia spp. was the most frequently detected fungus(7.89%). The genome sequencing assay was able to detect 5 rare pathogens including Alternaria pipionipisi, Ralstonia pickettii and difficult-to-culture organisms such as Orientia tsutsugamushi. Out of 35 samples, 25.85% samples were found to have co-infections. Out of 9 Antimicrobial resistant genes(ARGs) included in the panel, 6 ARGs were detected in the clinical samples. Most frequently detected ARGs include blaNDM, aph (3’), blaTEM, and blaSHV. 87.5% of Acinetobacter spp detected had aph (3’) and blaNDM and 25% had more than two ARGs. 66.6% of Escherichia coli had aph (3’) gene. DISCUSSIONS: Routine implementation of the genome sequencing assay enabled the comprehensive detection of different classes of pathogens from a range of clinical samples on a single platform The genome sequencing assay enabled the detection of underdiagnosed diseases such as scrub typhus and infections by rare pathogens. The study thus underscores the importance of performing routine genome sequencing assays as diagnostic solutions for patients with suspected infection.