应用微滴式数字PCR技术快速诊断新生儿侵袭性真菌病
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摘要
目的探讨微滴式数字PCR(ddPCR)技术在快速、精确诊断新生儿侵袭性真菌感染(IFI)中的临床应用价值。方法选择真菌高度保守序列18S rRNA为目标序列,通过引物设计,建立ddPCR检测真菌体系。在深圳一家医院新生儿重症监护室采集了有IFI高危因素和/或临床症状的83例患者血样(n=83),采用血培养及ddPCR法分别对血样进行真菌检测。结果 ddPCR检测真菌体系的特异性为100%,灵敏度可以达到3.2 copies/μL,重复性良好。临床试验中,22例确诊/临床诊断IFI患儿血样中,ddPCR检测阳性19例。61例拟诊/非IFI患儿血样中,ddPCR法检测结果有2例阳性。结论初步证明ddPCR技术可用于新生儿IFI的检测,该技术是新生儿IFI筛查甚至诊断很有前途的工具。
Objective To evaluate the clinical value of droplet digital PCR(ddPCR)in rapid and accurate diagnosis of invasive fungal infection(IFI)in neonates.Methods The highly conserved sequence of fungi 18S RNA was selected as the target sequence,and primers were designed to establish a ddPCR fungal detection system.Blood samples were collected from 83 neonates with high-risk factors for IFI and/or related clinical symptoms in the neonatal intensive care unit(NICU)of a hospital in Shenzhen,China.Blood culture and ddPCR were used for fungal detection.Results The ddPCR fungal detection system had a specificity of 100%and a sensitivity of 3.2 copies/μL,and had a good reproducibility.Among the 22 blood samples from neonates with a confirmed or clinical diagnosis of IFI,19 were detected positive by ddPCR.Among the 61 blood samples from neonates who were suspected of IFI or had no IFI,2were detected positive by ddPCR.Conclusions The ddPCR technique can be used for the detection of neonatal IFI and is a promising tool for the screening and even diagnosis of neonatal IFI.
Objective To evaluate the clinical value of droplet digital PCR(ddPCR)in rapid and accurate diagnosis of invasive fungal infection(IFI)in neonates.Methods The highly conserved sequence of fungi 18S RNA was selected as the target sequence,and primers were designed to establish a ddPCR fungal detection system.Blood samples were collected from 83 neonates with high-risk factors for IFI and/or related clinical symptoms in the neonatal intensive care unit(NICU)of a hospital in Shenzhen,China.Blood culture and ddPCR were used for fungal detection.Results The ddPCR fungal detection system had a specificity of 100%and a sensitivity of 3.2 copies/μL,and had a good reproducibility.Among the 22 blood samples from neonates with a confirmed or clinical diagnosis of IFI,19 were detected positive by ddPCR.Among the 61 blood samples from neonates who were suspected of IFI or had no IFI,2were detected positive by ddPCR.Conclusions The ddPCR technique can be used for the detection of neonatal IFI and is a promising tool for the screening and even diagnosis of neonatal IFI.
引文
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[16]De Pauw B,Walsh TJ,Donnelly JP,et al.Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group(EORTC/MSG)Consensus Group[J].Clin Infect Dis,2008,46(12):1813-1821.
[17]中国侵袭性真菌感染工作组.血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则(第五次修订版)[J].中华内科杂志,2017,56(6):453-459.
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[20]Mennink-Kersten MA,Verweij PE.Non-culture-based diagnostics for opportunistic fungi[J].Infect Dis Clin North Am,2006,20(3):711-727.
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[22]Halliday CL,Kidd SE,Sorrell TC,et al.Molecular diagnostic methods for invasive fungal disease:the horizon draws nearer?[J].Pathology,2015,47(3):257-269.
[2]Jiang X,Dong D,Bian L,et al.Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples[J].Front Microbiol,2016,7:916.
[3]Roilides E.Invasive Candidiasis in neonates and children[J].Early Human Development,2011,87:S75-S76.
[4]Gupta P,Ahmad A,Khare V,et al.Comparative evaluation of pan-fungal real-time PCR,galactomannan and(1-3)-beta-D-glucan assay for invasive fungal infection in paediatric cancer patients[J].Mycoses,2017,60(4):234-240.
[5]Cahan H,Deville JG.Outcomes of neonatal Candidiasis:the impact of delayed initiation of antifungal therapy[J].Int JPediatr,2011,2011:813871.
[6]唐仕芳,陈龙,赵锦宁,等.新生儿重症监护病房侵袭性真菌感染易感因素及早期诊断探讨[J].临床儿科杂志,2011,29(9):810-812.
[7]Valero C,de la Cruz-Villar L,Zaragoza O,et al.New panfungal real-time PCR assay for diagnosis of invasive fungal infections[J].J Clin Microbiol,2016,54(12):2910-2918.
[8]Greenberg RG,Benjamin DJ.Neonatal Candidiasis:diagnosis,prevention,and treatment[J].J Infect,2014,69 Suppl 1:S19-S22.
[9]Pinheiro LB,Coleman VA,Hindson CM,et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Anal Chem,2012,84(2):1003-1011.
[10]Hindson BJ,Ness KD,Masquelier DA,et al.High-throughput droplet digital PCR system for absolute quantitation of DNAcopy number[J].Anal Chem,2011,83(22):8604-8610.
[11]Persaud D,Gay H,Ziemniak C,et al.Absence of detectable HIV-1 viremia after treatment cessation in an infant[J].N Engl JMed,2013,369(19):1828-1835.
[12]Sedlak RH,Cook L,Huang ML,et al.Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR[J].Clin Chem,2014,60(5):765-772.
[13]Ramos JT,Villar S,Bouza E,et al.Performance of a quantitative PCR-based assay and beta-d-glucan detection for diagnosis of invasive Candidiasis in very-low-birth-weight preterm neonatal patients(CANDINEO Study)[J].J Clin Microbiol,2017,55(9):2752-2764.
[14]Tang XL,Hua Y,Guan Q,et al.Improved detection of deeply invasive candidiasis with DNA aptamers specific binding to(1→3)-beta-D-glucans from Candida albicans[J].Eur J Clin Microbiol Infect Dis,2016,35(4):587-595.
[15]陈超.新生儿真菌感染的诊治[J].中国实用儿科杂志,2011,26(1):3-6.
[16]De Pauw B,Walsh TJ,Donnelly JP,et al.Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group(EORTC/MSG)Consensus Group[J].Clin Infect Dis,2008,46(12):1813-1821.
[17]中国侵袭性真菌感染工作组.血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则(第五次修订版)[J].中华内科杂志,2017,56(6):453-459.
[18]Reissa E,Lasker BA,Iqbal NJ,et al.Molecular epidemiology of Candida parapsilosis sepsis from outbreak investigations in neonatal intensive care units[J].Infect Genet Evol,2008,8(2):103-109.
[19]Ting J,Roberts A,Synnes A,et al.Invasive fungal infections in neonates in Canada:epidemiology and outcomes[J].Pediatr Infect Dis J,2018,37(11):1154-1159.
[20]Mennink-Kersten MA,Verweij PE.Non-culture-based diagnostics for opportunistic fungi[J].Infect Dis Clin North Am,2006,20(3):711-727.
[21]Khot PD,Fredricks DN.PCR-based diagnosis of human fungal infections[J].Expert Rev Anti Infect Ther,2009,7(10):1201-1221.
[22]Halliday CL,Kidd SE,Sorrell TC,et al.Molecular diagnostic methods for invasive fungal disease:the horizon draws nearer?[J].Pathology,2015,47(3):257-269.