scholarly journals Multicentre evaluation of two multiplex PCR platforms for the rapid microbiological investigation of nosocomial pneumonia in UK ICUs: the INHALE WP1 study

2020 ◽  
Author(s):  
Virve I Enne ◽  
Alp Aydin ◽  
Rossella Baldan ◽  
Dewi R Owen ◽  
Hollian Richardson ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Latent Class ◽  
Controlled Trial ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Icu Patients ◽  
Microbiological Investigation ◽  
Antimicrobial Resistance Genes ◽  
Tract Infections

SummaryBackgroundICU patients with hospital-acquired or ventilator-associated pneumonia (HAP or VAP) have high mortality, so broad-spectrum antibiotics are initiated at clinical diagnosis, then refined after 2-3 days, once microbiology results become available. Unfortunately, culture-based microbiological investigation is also insensitive, with aetiological agents remaining unidentified in many cases. This leads to extended over-treatment of patients with susceptible pathogens, whilst those with highly-resistant pathogens are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship. The INHALE study compared two PCR platforms for HAP/VAP diagnosis against routine microbiology (RM), identifying one to progress into a Randomised Controlled Trial (RCT).MethodsSurplus routine sputa, endotracheal tube exudates and bronchoalveolar lavages were collected from ICU patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Samples were tested (or frozen for testing) within 72h of collection. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Agreement between machine- and RM-results was categorised as ‘full positive/negative concordance’, ‘partial concordance’ or ‘major/minor discordance’. Bayesian latent class (BLC) analysis was used to estimate the sensitivity and specificity of each test, incorporating information from both PCR panels, 16S rDNA analysis and RM.FindingsIn 652 eligible samples; PCR identified pathogens in considerably more samples compared with RM: 60.4% and 74.2% for Unyvero and FilmArray respectively vs. 44.2%. Both tests also recorded more organisms per sample than routine culture, with the two PCR tests frequently in agreement with each other. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7-100.0% and specificity of 87.5-99.5%; Unyvero had sensitivity of 83.3-100.0%% except for Klebsiella aerogenes (50.0%) and Serratia marcescens (77.8%), and specificity of 89.4-99.0%. BLC analysis indicated that, compared with PCR, RM had low sensitivity, ranging from 27.0% to 69.4% for common respiratory pathogens. PCR detected more high-consequence antimicrobial resistance genes than would have been predicted by RM and susceptibility testing; around half the host strains could be detected when culture was repeated and they were sought assiduously.InterpretationConventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than RM, detecting potential pathogens in patient samples reported as culture negative. FilmArray had slightly higher sensitivity than Unyvero for common pathogens and was chosen for INHALE’s RCT, based on the balance of these results, a swifter turnaround time (75 min vs. 6h), and a smaller footprint. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

scholarly journals Multicentre evaluation of two multiplex PCR platforms for the rapid microbiological investigation of nosocomial pneumonia in UK ICUs: the INHALE WP1 study

Thorax ◽  
2022 ◽  
pp. thoraxjnl-2021-216990
Author(s):  
Virve I Enne ◽  
Alp Aydin ◽  
Rossella Baldan ◽  
Dewi R Owen ◽  
Hollian Richardson ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Latent Class ◽  
Antimicrobial Treatment ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
Microbiological Investigation ◽  
Increased Sensitivity ◽  
Low Sensitivity ◽  
Tract Infections

BackgroundCulture-based microbiological investigation of hospital-acquired or ventilator-associated pneumonia (HAP or VAP) is insensitive, with aetiological agents often unidentified. This can lead to excess antimicrobial treatment of patients with susceptible pathogens, while those with resistant bacteria are treated inadequately for prolonged periods. Using PCR to seek pathogens and their resistance genes directly from clinical samples may improve therapy and stewardship.MethodsSurplus routine lower respiratory tract samples were collected from intensive care unit patients about to receive new or changed antibiotics for hospital-onset lower respiratory tract infections at 15 UK hospitals. Testing was performed using the BioFire FilmArray Pneumonia Panel (bioMérieux) and Unyvero Pneumonia Panel (Curetis). Concordance analysis compared machine and routine microbiology results, while Bayesian latent class (BLC) analysis estimated the sensitivity and specificity of each test, incorporating information from both PCR panels and routine microbiology.FindingsIn 652 eligible samples; PCR identified pathogens in considerably more samples compared with routine microbiology: 60.4% and 74.2% for Unyvero and FilmArray respectively vs 44.2% by routine microbiology. PCR tests also detected more pathogens per sample than routine microbiology. For common HAP/VAP pathogens, FilmArray had sensitivity of 91.7%–100.0% and specificity of 87.5%–99.5%; Unyvero had sensitivity of 50.0%–100.0%%, and specificity of 89.4%–99.0%. BLC analysis indicated that, compared with PCR, routine microbiology had low sensitivity, ranging from 27.0% to 69.4%.InterpretationConventional and BLC analysis demonstrated that both platforms performed similarly and were considerably more sensitive than routine microbiology, detecting potential pathogens in patient samples reported as culture negative. The increased sensitivity of detection realised by PCR offers potential for improved antimicrobial prescribing.

scholarly journals Rapid Multiplex Testing for Upper Respiratory Pathogens in the Emergency Department: A Randomized Controlled Trial

2019 ◽  
Author(s):  
Larissa May ◽  
Grant Tatro ◽  
Eduard Poltavskiy ◽  
Benjamin Mooso ◽  
Simson Hon ◽  
...  
Keyword(s):  
Emergency Department ◽  
Length Of Stay ◽  
Usual Care ◽  
Controlled Trial ◽  
Antibiotic Use ◽  
Respiratory Pathogens ◽  
Upper Respiratory Tract ◽  
Upper Respiratory ◽  
Tract Infections ◽  
The Impact

Abstract Background Acute upper respiratory tract infections are a common cause of Emergency Department (ED) visits and often result in unnecessary antibiotic treatment.  Methods We conducted a randomized clinical trial to evaluate the impact of a rapid, multi-pathogen respiratory panel (RP) test versus usual care (control). Patients were eligible if they were ≥12 months old, had symptoms of upper respiratory infection or influenza like illness, and were not on antibiotics. The primary outcome was antibiotic prescription; secondary outcomes included antiviral prescription, disposition, and length of stay (ClinicalTrials.gov# NCT02957136). Results Of 191 patients enrolled, 93 (49%) received RP testing; 98 (51%) received usual care. Fifty-three (57%) RP and 7 (7%) control patients had a virus detected and reported during the ED visit (p=0.0001). Twenty (22%) RP patients and 33 (34%) usual care patients received antibiotics during the ED visit (-12% [95% CI -25%, 0.4%]; p=0.06/0.08); 9 RP patients received antibiotics despite having a virus detected. The magnitude of antibiotic reduction was greater in children (-19%) versus adults (-9%; post-hoc analysis). There was no difference in antiviral use, length of stay, or disposition. Conclusions Rapid RP testing was associated with a trend towards decreased antibiotic use, suggesting a potential benefit from more rapid viral tests in the ED. Future studies should determine if specific groups are more likely to benefit from testing and evaluate relative cost and effectiveness of broad testing, focused testing, and a combined diagnostic and antimicrobial stewardship approach.

scholarly journals Phenotypic and Molecular Detection of Resistance Genes from Multi-drug Resistant Escherichia coli Isolated from Patients Attending Selected Hospitals in Damaturu, Yobe State, Nigeria

2021 ◽  
Vol 8 (9) ◽  
pp. 396-407
Author(s):  
Sheriff Wakil ◽  
Mustafa Alhaji Isa ◽  
Adam Mustapa
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Resistance Genes ◽  
Molecular Detection ◽  
Multidrug Resistant ◽  
Clinical Samples ◽  
E Coli ◽  
Resistant Genes ◽  
Tract Infections

Multidrug resistance among Escherichia coli causing urinary tract infections (UTIs) and diarrhea are major public health problem worldwide which cause difficulty in treating the infections caused by Escherichia coli due to the high resistances. The study is aimed to determine the phenotypic and molecular detection of multidrug resistant E. coli isolated from clinical samples of patients attending selected Hospitals in Damaturu, Yobe State-Nigeria. Methods: Two hundred (200) clinical samples were collected aseptically from patient diagnosed with (100 stool samples) and UTI’s (100 urine samples) using sterile universal container. The samples were processed using standard microbiological methods for identification of E. coli. Samples were cultured on MacConkey agar (stool) and Cystine lactose electrolyte deficient agar (urine). The resulting colonies of isolates were further subculture on Eosin methylene blue agar for confirmatory and followed by gram stain, biochemical identification at Microbiology laboratory unit of Yobe State Specialist and Yobe State Teaching Hospital respectively. The antimicrobial susceptibility patterns were determined using Kirby-Bauer disc diffusion techniques and the phenotypic expression of extended spectrum beta-lactamases (ESBLs) were determined using modified double disc synergy test (MDDST) and also the three (3) resistance genes (blaTEM, accC1 and qnrA) were detected using polymerase chain reaction. Results: One hundred and twenty-two (122) isolates were resistant to antibiotics. The highest level of resistance was against amoxicillin (90.2%) while the least resistance was against sparfloxacin (24.3%). Thirty-seven (37) E. coli isolates shows MDR; the highest MDR was (24.3%) while least MDR was (5.4%). The PCR amplification of resistant genes (blaTEM, accC1 and qnrA) were detected on E. coli that shows positive ESBL and the bands were separated using agarose gel electrophoresis. Conclusion: The findings of this study show augmentin, ciprofloxacin and sparfloxacin are the most effective antibiotics against E. coli isolated from patients attending the two hospitals in Damaturu; who are diagnose with UTI and diarrheic infection. The resistant genes include; blaTEM, accC1 and qnrA coding for beta-lactam, aminoglycoside and quinolones were present in E. coli isolated from patients attending selected Hospitals in Yobe State, Nigeria. Keywords: Multidrug resistant, Escherichia coli, extended spectrum beta lactamase, resistance-associated genes, urinary tract infections, diarrheic.

scholarly journals Development and application of a method to detect 27 respiratory pathogens using multiplex RT-PCR combined with MassARRAY technology

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively. Objectives To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value. Methods Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR. Results The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/μl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100, 98.68, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test). Conclusion Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

scholarly journals 1207. Acquisition and Quantification of Antimicrobial Resistance Genes in the Gut Microbiome of Ugandan Women Exposed to Small-Scale Chicken Farming

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S366-S366
Author(s):  
Meti D Debela ◽  
Daniel M Muyanja ◽  
Bernard Kakuhikire ◽  
Charles Baguma ◽  
David R Bangsberg ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Controlled Trial ◽  
Intervention Group ◽  
Fluoroquinolone Resistance ◽  
Small Scale ◽  
Class A ◽  
Antimicrobial Resistance Genes ◽  
Wide Range ◽  
One Year

Abstract Background Antibiotic use in livestock farming is thought to be a major contributor to the spread of antimicrobial resistance (AMR) genes in humans. However, quantitative data in this in this field are rare. To address this gap in the literature, we examined the prevalence of clinically important AMR genes before and after the introduction of chicken farming among women in rural Uganda. Methods We recruited a subset of women participating in a waitlist-randomized controlled trial of small-scale hybrid chicken farming in rural Uganda. Tetracycline is routinely administered to chicks during brooding. Stool samples before and one year after chicken introduction were obtained from six women randomized to the control arm, from five women randomized to the intervention arm, and from chickens. Microbial DNA was extracted from chicken and human stool and screened for 87 AMR genes using validated qPCR arrays (Qiagen). Results The median age was 35 years. At baseline, 10 of the women reported animal contact, most commonly goats (n = 8), free ranging village chickens (n = 7), cats (n = 4), and dogs (n = 4). During baseline testing of the women’s stool, we detected 18 genes conferring AMR to aminoglycosides, fluoroquinolones, macrolides, lincosamides, streptogramin B, Class A-C β-lactamases and tetracycline efflux pumps. Chickens harbored 23 AMR genes from the same classes as found in humans, and were also found to have vancomycin resistance genes (Van B and C) and Group D β-lactamases (OXA-58 and OXA-10). At one year, six new AMR genes emerged in controls, including one present in chickens; CTX-M-1, a Class A β-lactamase. In contrast, seven new AMR genes emerged in the intervention group, including four present in chickens: SHV, SHV(238G240E), (Class A β lactamases) and QnrS, QnrB-5 (fluoroquinolone resistance genes). Two AMR genes gained by both control and intervention groups were not present in chickens. Conclusion Women exposed to small-scale chicken farming acquired more AMR genes compared with unexposed participants. Chickens harbored many of the genes that emerged in humans. Introduction of antibiotic-treated animals may result in the transfer of AMR genes from animals to humans, even among humans exposed to a wide range of animals at baseline. Disclosures All authors: No reported disclosures.

scholarly journals Detection of Antimicrobial Resistance Genes Associated with Carbapenem Resistance from the Whole-Genome Sequence of Acinetobacter baumannii Isolates from Malaysia

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Mohan Rao ◽  
Fairuz A. Rashid ◽  
Surianti Shukor ◽  
Rohaidah Hashim ◽  
Norazah Ahmad
Keyword(s):  
Resistance Genes ◽  
Clonal Complex ◽  
Clinical Sample ◽  
Gene Families ◽  
Whole Genome Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Carbapenem Resistant ◽  
Antimicrobial Resistance Genes ◽  
Carbapenemase Gene

Background. The spread of carbapenem-resistant A. baumannii (CrAb) is gaining worldwide attention. The spread of this pathogen is largely due to its ability to acquire various resistance genes of intrinsic and extrinsic origins that confer unpredictable susceptibility to β-lactams. The aim of this study was to analyze β-lactamase genetic compositions of CrAb in Malaysia. Methods. Whole-genome sequencing (WGS) was carried out on 13 CrAb isolates from clinical samples in Malaysia from 2011 to 2016. Results. Endotracheal aspirate was the dominant clinical sample source (n = 6), and only one isolate was obtained from wound swab. A total of 6 sequence types (STs) of the Oxford scheme were identified, including 4 reported STs and 2 novel STs. Eleven isolates were classified into clonal complex 92 (CC92/ICII), among which ST195 and ST208 were the most prevalent STs. All 13 CrAb isolates harbored multiple β-lactamase genes. blaOXA-23 (n = 13) and blaOXA-66 (n = 11) were the dominant carbapenemase gene families found in these isolates. All isolates harbor blaADC, blaOXA-51-like, and blaOXA-23-like genes. blaTEM (n = 7), blaNDM-1 (n = 3), blaCARB-8 (n = 1), and blaPER-3 (n = 1) are amongst other β-lactamase genes found in this study. ISAba1 was found upstream to blaOXA-23 (n = 13), blaOXA-66 (n = 1), and blaADC (n = 11). All blaNDM-1 isolates had ISAba125 (mobile genetic element) upstream to the genes. All isolates were positive for Tn2006/2008 and Tn2009 but were negative for Tn2007. Conclusion. Most of the isolates were grouped under the CC92 clonal complex which belongs to international clonal lineage 2. These findings predict that carriage of carbapenem-resistant genes possibly constitutes the underlying basis of high level of international clone II prevalence. Therefore, molecular surveillance and antimicrobial stewardship are essential in implementing policies to prevent and control the spread of CrAb in hospital settings.

Evolution and genomic insight into methicillin-resistant Staphylococcus aureus ST9 in China

2021 ◽  
Author(s):  
Nansong Jiang ◽  
Kelly L Wyres ◽  
Jun Li ◽  
Andrea T Feßler ◽  
Henrike Krüger ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Resistance Genes ◽  
Antimicrobial Agents ◽  
Complex Structure ◽  
Rapid Evolution ◽  
Clinical Samples ◽  
Chromosomal Dna ◽  
Genomic Epidemiology ◽  
Antimicrobial Resistance Genes ◽  
Bayesian Phylogeny

Abstract Objectives To reconstruct the evolutionary history and genomic epidemiology of Staphylococcus aureus ST9 in China. Methods Using WGS analysis, we described the phylogeny of 131 S. aureus ST9 isolates collected between 2002 and 2016 from 11 provinces in China, including six clinical samples from Taiwan. We also investigated the complex structure and distribution of the lsa(E)-carrying multiresistance gene cluster, and genotyped prophages in the genomes of the ST9 isolates. Results ST9 was subdivided into one major (n = 122) and one minor (n = 9) clade. Bayesian phylogeny predicted the divergence of ST9 isolates in pig farming in China as early as 1987, which then evolved rapidly in the following three decades. ST9 isolates shared similar multiresistance properties, which were likely acquired before the ST9 emergence in China. The accessory genome is highly conserved, and ST9 harboured similar sets of phages, but lacked certain virulence genes. Conclusions Host exchange and regional transmission of ST9 have occurred between pigs and humans. Pig rearing and trading might have favoured gene exchanges between ST9 isolates. Resistance genes, obtained from the environment and other isolates, were stably integrated into the chromosomal DNA. The abundance of resistance genes among ST9 is likely attributed to the extensive use of antimicrobial agents in livestock. Phages are present in the genomes of ST9 and may play a role in the rapid evolution of this ST. Although human ST9 infections are rare, ST9 isolates may constitute a potential risk to public health as a repository of antimicrobial resistance genes.

scholarly journals From the Urinary Catheter to the Prevalence of Three Classes of Integrons, β-Lactamase Genes, and Differences in Antimicrobial Susceptibility of Proteus mirabilis and Clonal Relatedness with Rep-PCR

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Arezoo Mirzaei ◽  
Bahram Nasr Esfahani ◽  
Abbasali Raz ◽  
Mustafa Ghanadian ◽  
Sharareh Moghim
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antimicrobial Susceptibility ◽  
Proteus Mirabilis ◽  
Diffusion Method ◽  
Intrinsic Resistance ◽  
Microtiter Plate Assay ◽  
Esbl Gene ◽  
Antimicrobial Resistance Genes ◽  
Tract Infections

Introduction. Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. Method. Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (intI1, intI2, intI3, blaTEM, blaCTX-M, blaSHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR. Result. From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was blaTEM. Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns. Conclusion. In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The intI1 and blaTEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future.

scholarly journals Development and Application of a Method to Detect 27 Respiratory Pathogens Using Multiplex RT-PCR Combined with MassARRAY Technology

2021 ◽  
Author(s):  
Huan Zhao ◽  
Yichao Yang ◽  
Jiangfeng Lyu ◽  
Xuyi Ren ◽  
Wei Cheng
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Early Recognition ◽  
Detection System ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Tract Infections

Abstract Background: Respiratory tract infections are the most common infections that lead to morbidity and mortality worldwide. Early recognition and precise diagnosis of microbial etiology is important to treat LRTIs promptly, specifically and effectively.Objectives: To establish a method based on multiplex reverse transcription (MRT)-PCR and MassARRAY technology for the simultaneous detection of 27 respiratory pathogens and explore its clinical application value.Methods: Analytical sensitivity and specificity of the MRT-PCR-MassARRAY system were validated using inactivated bacterial and viral strains. Also we analyzed samples from 207 patients by MassARRAY methods and compared the results with consensus PCR/reverse transcription (RT)-PCR.Results: The minimum detection limit of our MRT-PCR-MassARRAY method for pathogens was 10–100 copies/µl, with high specificity. Comparison test with consensus PCR/RT-PCR on 207 clinical samples, the positive, negative, and total correlation rates were 100%, 98.68%, and 99.03%, respectively. There was a high degree of agreement between the test results of the two methods (P < 0.01 by McNemar’s test).Conclusion: Our detection system of 27 respiratory pathogens based on MassARRAY technology has high sensitivity and specificity, high throughput, and is simple to operate. It provides diagnostic value for the clinical diagnosis of respiratory pathogens and is of great significance in the screening of respiratory pathogens.

scholarly journals Combined nanopore adaptive sequencing and enzyme-based host depletion efficiently enriched microbial sequences and identified missing respiratory pathogens

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Mingyu Gan ◽  
Bingbing Wu ◽  
Gangfeng Yan ◽  
Gang Li ◽  
Li Sun ◽  
...  
Keyword(s):  
Standard Method ◽  
Respiratory Tract Infections ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Interquartile Range ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Combined Method ◽  
Tract Infections ◽  
Enrichment Efficiency

Abstract Background Enzyme-based host depletion significantly improves the sensitivity of clinical metagenomics. Recent studies found that real-time adaptive sequencing of DNA molecules was achieved using a nanopore sequencing machine, which enabled effective enrichment of microbial sequences. However, few studies have compared the enzyme-based host depletion and nanopore adaptive sequencing for microbial enrichment efficiency. Results To compare the host depletion and microbial enrichment efficiency of enzyme-based and adaptive sequencing methods, the present study collected clinical samples from eight children with respiratory tract infections. The same respiratory samples were subjected to standard methods, adaptive sequencing methods, enzyme-based host depletion methods, and the combination of adaptive sequencing and enzyme-based host depletion methods. We compared the host depletion efficiency, microbial enrichment efficiency, and pathogenic microorganisms detected between the four methods. We found that adaptive sequencing, enzyme-based host depletion and the combined methods significantly enriched the microbial sequences and significantly increased the diversity of microorganisms (p value < 0.001 for each method compared to standard). The highest microbial enrichment efficiency was achieved using the combined method. Compared to the standard method, the combined method increased the microbial reads by a median of 113.41-fold (interquartile range 23.32–327.72, maximum 1812), and the number of genera by a median of 70-fold (interquartile range 56.75–86.75, maximum 164). The combined method detected 6 pathogens in 4 samples with a median read of 547, compared to 5 pathogens in 4 samples with a median read of 4 using the standard method. Conclusion The combined method is an effective, easy-to-run method for enriching microbial sequences in clinical metagenomics from sputum and bronchoalveolar lavage fluid samples and may improve the sensitivity of clinical metagenomics for other host-derived clinical samples.

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