Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR
Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR
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DOI：
10.1128/jcm.01523-14
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发表时间：
2014-10-01
影响因子：
9.4
通讯作者：
Falsey, Ann R.
中科院分区：
文献类型：
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作者：
Branche, Angela R.;Walsh, Edward E.;Falsey, Ann R.
呼吸道感染（RTI）经常导致成人住院。鼻咽拭子（NTS）的诊断性病毒逆转录酶PCR （RT-PCR）通过告知抗病毒药物的使用和适当的隔离，对患者护理有用。然而，自动RT-PCR系统不适合利用痰液，因为它的粘度。我们评估了一种在全自动呼吸道病毒面板RT-PCR检测（FilmArray）中处理痰样本的简单方法。对2008-2012年住院成人RTI患者收集的存档痰液和NTS样本进行评估。选取了10种常见病毒单效RT-PCR阳性的痰样本子集。将无菌棉签浸泡在痰液中，在700 μ L无菌水中旋转（浸泡和旋转法），并进行FilmArray检测。对“浸泡”的痰液和NTS样本进行定量RT-PCR检测甲型流感（Flu A）、呼吸道合胞病毒（RSV）、冠状病毒OC43 （OC43）和人偏肺病毒（HMPV）。在965种疾病中，使用单链RT-PCR鉴定出了31%的病毒。105名受试者中只有痰液样本呈阳性，其中35%（22/64）为流感病例，且显著提高了NTS单独诊断的检出率（302/965[31%]对197/965 [20%]，P = 0.0001）。FilmArray试验采用灌篮和旋流法评估的108份痰样本中，99份（92%）呈阳性。定量RT-PCR显示，与NTS样本相比，浸泡的痰样本中甲型流感、RSV和HMPV的平均病毒载量更高（P = 0.0001, P = 0.006和P = 0.011）。浸泡旋流法是一种在全自动PCR系统中可靠处理痰样本的简单实用方法。痰液中较高的病毒载量可能比单独的NTS检测增加检测。
Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 mu L of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on "dunked" sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone.