| BackgroundGlobally,respiratory infections are a major contributor to the amount of illness,and are a predominant cause of fatalities in children.A speedy identification of the causative agents of respiratory illnesses can shorten the duration of hospitalization,ensure proper antibiotic management,and forestall hospital-acquired infections.The integration of coding microspheres(particles),laser technology,flow cytometry technology,and digital signal processing technology,commonly referred to as liquid chip or suspension array technology,is a high-throughput and multi-target detection tool which is employed in a wide range of fields,including biomarker research,drug and vaccine development,transplant medicine,infectious diseases and personalized tumor treatment.The Luminex xTAG platform is the first of its kind,utilizing the same sized and differently stained microspheres for protein and nucleic acid multiplex analysis.Its xTAG oligonucleotide tag technology was used to develop the NxTAG(?)RPP reagent,which allows for the simultaneous detection of 21 different types of respiratory viruses and bacteria.Compared to the first generation of products,PCR and hybridization have been combined into one step,resulting in a reduction in detection turnaround time(TAT)to 4 hours.Since 2001,Human metapneumovirus(hMPV),a virus from the paramyxoviridae family,has been identified as a cause of respiratory illness in infants and children globally,being a new member of the subspecies of pneumonia viruses.Humans infected with metapneumovirus typically experience respiratory-related symptoms such as fever,cough,and shortness of breath.In some cases,the virus can cause severe pneumonia and neurological problems,raising alarm in society.Over the last 10 years,hMPV has become increasingly widespread in China,and with advances in molecular biology,the disease has been monitored more closely in different areas.Purposes1.We implemented standard laboratory practices,including first-generation sequencing,virus isolation and culture,and real-time PCR of respiratory viruses,to comprehensively evaluate the effectiveness of NxTAG(?)RPP in detecting domestic epidemic respiratory pathogens,and to provide data for its clinical application.2.Between January 2010 and June 2019,our hospital gathered data on 4655 cases of respiratory infections,then used real-time PCR to look for human metapneumovirus RNA in the nasal swabs,and subsequently amplified the F gene sequence of the virus through RT-PCR and Sanger sequencing,before carrying out genotyping and phylogenetic analysis through gene sequence alignment.Method1.Between November 2016 and July 2018,492 nasopharyngeal swab specimens from patients with respiratory infection at Zhujiang Hospital were evaluated using Nx TAG(?)RPP,RT-PCR coupled with Sanger sequencing,real-time PCR for detection of 18 lab-developed respiratory pathogens,colloidal gold immunochromatography for c hlamydia pneumoniae and legionella pneumophila,and isolation and culture for influ enza A and B virus.The accuracy of RPP detection and consistency with real-time P CR for the respiratory pathogens commonly used in labs was evaluated.2.Between January 2010 and June 2019,our hospital kept records of 4655 cases of respiratory infections,and the presence of human metapneumovirus RNA in the nasal swab specimens of the patients was established by real-time PCR technology.RT-PCR and Sanger sequencing techniques were used to amplify the F gene sequences of human metapneumovirus,and gene sequence alignment was conducted to conduct genotyping and phylogenetic analysis.Results1.The comparison between NxTAG(?)RPP and sequencing for the detection of 17 respiratory pathogen specimens revealed outstanding concordance,with kappa values ranging from 0.9629 to 1.0000.The NxTAG(?)RPP had impressive results,usually exhibiting a sensitivity of more than 93.75%and a specificity of 98.69%or higher.Using first-generation sequencing as a benchmark,the sensitivity of the NxTAG(?)RPP test for respiratory pathogens is 100%,and it has a specificity of over 99%for most other pathogens,except for HBoV(93.75%)and ADV(97.37%).The NxTAG(?)RPP was found to be 98.8%accurate(95%CI:96.9-99.6%),and its precision was 100%for the majority of pathogens when compared to the laboratory’s self-constructed real-time fluorescence RT-PCR.The NxTAG(?)RPP test had an overall accuracy of 99.4%(95%CI:99.1-99.5%),and the majority of pathogens were detected with a specificity of greater than 98.0%.The percentage of agreement between NxTAG(?)RPP and FluA-H1 culture was 43.24%,and the percentage of agreement between NxTAG(?)RPP and FluB culture was 64.71%,which was determined after isolating and culturing influenza A and B viruses.2.Between January 2010 and June 2019,our hospital observed a 5.05%detection rate of hMPV among all respiratory viral infections,with the majority of cases occurring in children aged 4 or younger,men being more likely to be infected,and the most common symptoms of the upper respiratory tract infection being fever,cough,and sputum production.In the last few years,the hMPV virus has been most active from February to May,with noticeable fluctuations throughout the seasons.There is little variation in the demographic and clinical characteristics of infected children.The majority of cases(34.49%,74/235)showed evidence of multiple viral infections,particularly of Rhv,RSVA,or ADV,but no pattern of co-infection was identified.Out of the 155 positive samples,118 were correctly identified,with 41(34.74%)of them being of A2 type,60(50.85%)of them being of B1 type,and 17(14.41%)of them being of B2 type.In this investigation,no A1 type was present,and of the 41 A2 samples,2 were of the A2a variety,37 were of the A2b type,and 2 plants showed characteristics that leaned toward A1.Throughout the study period,two types of human metapneumovirus,A and B,were present,with one strain taking precedence in any given year,and the prevailing strain changing in 2013/2014,2015/2016,and 2018/2019.The phylogenetic comparison between the human metapneumovirus circulating in the region and the reference strain show that they are closely related,yet the B1 type can be categorized into two distinct groups in 2015/2016,implying that the B1 subtype has possibly mutated and evolved to better suit the environment.ConclusionsThis research illustrates the impressive accuracy and precision of NxTAG(?)RPP in the detection of 21 respiratory pathogens,as well as its clear advantage in terms of multiplex detection speed and output,making it an ideal choice for clinical labs to screen for common acute respiratory infection pathogens.In this region,Human metapneumovirus is a major cause of acute respiratory illnesses in children aged 4 and younger,and both type A and B of the virus circulate simultaneously with the dominant strain changing periodically. |
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