Application And Evaluation Of Nanopore Targeted Sequencing In The Detection Of Pathogen

Objective Infectious diseases are the leading cause of death in patients.Characterized by long turnaroud time and low sensitivity,traditional microbial detection approaches are prone to misdiagnosis,making it difficult to rational use of anti-infective drugs at early stages of infections.Nanopore Targeted Sequencing(NTS),which is a representative of third-generation sequencing technologies,have been used for microbial detection.The aim of this study was to comprehensively assess the clinical utility of NTS for diagnosis of infectious diseases through retrospective and prospective studies.Methods In the first part of the research,472 clinical specimens from 436 patients hospitalized at the Central South Hospital of Wuhan University from March 2021 to May 2021 were subjected to NTS and culture identification.Six types of specimens were tested by NTS,including 160 bronchoalveolar lavage fluid(BALF)specimens,121 blood specimens,74 urine specimens,50 pleural and ascites fluid samples,42 cerebrospinal fluid(CSF)specimens,and 25 wound secretion specimens.The clinical data,microbiological cultures and NTS results of the 436 patients included were retrospectively analyzed,while Sanger sequencing was used to validate the pathogens detected by NTS in order to objectively assess the clinical utility of NTS for diagnosis of infectious diseases.In the second part of this research,it is a prospective study including 50 CSF specimens from 50 patients with suspected CNS infections who were treated in the Department of Neurosurgery at Zhongnan Hospital of Wuhan University from July2021 to December 2021.The results of 50 CSF specimens by NTS were verified using NGS(next-generation sequencing)and culture in parallel.The results of 9specimens by NTS which weren't consistent with those by NGS and culture were further identified using 16 S r DNA amplification sequencing method.The demographic information and clinical manifestations of patients were collected,including age,sex,clinical diagnosis,and other laboratory results(blood routine,CSF routine,CSF biochemistry,procalcitonin,and interleukin-6 etc.).This study aimed to objectively assess the value of NTS for diagnosis of central nervous system infections.Results For the first part,the positivity rate for NTS was 86.7%(409/472),which was significantly higher than the positivity rate for microbial culture of 26.9%(127/472).the diagnostic sensitivity,specificity,positive predictive value and negative predictive value of NTS were 94.5%,31.8%,79.9% and 66.7%,respectively.The average validation rate of Sanger sequencing was 85.2% and varied widely among pathogen species,with the highest validation rate for Cryptococcal neoformans(100%)and the lowest validation rate for Burkholderia cepacia(0%).The validation rate for Sanger sequencing also varied with sample type,with the highest validation rate for pleural and ascites fluid samples(95.8%),followed by BALF(88.7%),urine(86.8%)and wound secretions(85.0%),and the lowest validation rate for CSF,which was only58.8%.For the second part of the paper,for the 50 patients with suspected CNS infection,the positivity rate for culture was 16.0%(8/50),the positivity rate for m NGS was 94.0%(47/50),and the positivity rate for NTS was 76.0%(38/50).The overall validation rate was 86.7%(52/60).The sensitivity and specificity were 71.0%and 15.8%,respectively,and the positive and negative predictive values were 57.9%and 25.0%,respectively.Conclusions NTS can accurately detect pathogenic bacteria in clinical samples with much higher sensitivity than traditional microbial culture.It can be effectively applied in the treatment of clinical infectious diseases to provide personalized care to patients.For CNS infections,NTS can replace high-throughput and high-cost m NGS in some cases,resulting in significant cost savings to the healthcare system,especially in patients with severe underlying disease,neurosurgery,human immunodeficiency virus infection,or immunocompromised organ transplantation.However,there are inevitable drawbacks including the inability of NTS to identify microbial activity and to provide information on microbial virulence and drug resistance due to targeted amplification.It has relatively limited discriminatory power when it comes to the identification of genera or higher taxa.What's more,it is highly susceptible to contamination by environmentally flora and normally colonizing bacteria.Accurate interpretation of sequencing data is critical for clinical diagnosis and treatment.