Department of Pathology, Stanford University School of Medicine, Stanford, California, United States.
Clinical Virology Laboratory, Stanford Health Care, California, United States.
J Clin Virol. 2021 Mar;136:104757. doi: 10.1016/j.jcv.2021.104757. Epub 2021 Feb 9.
Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available.
We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes.
The MeV triple-target rRT-PCR was tested against serial dilutions (7.0-2.0 log copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus.
The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions.
麻疹病毒（Measles virus，MeV）是全球范围内引起急性发热性疾病和儿童死亡的重要原因，近期美国的暴发与疫苗接种不足有关。MeV 具有高度传染性，及时诊断对于限制其传播至关重要。RNA 检测是急性麻疹诊断最敏感的方法；然而，MeV 核酸扩增检测方法并不广泛可用。
我们对同时检测 MeV N、H 和 L 基因的三重靶标实时 RT-PCR（rRT-PCR）检测方法进行了诊断准确性研究。
MeV 三重靶标 rRT-PCR 检测了代表循环基因型的五个 MeV 分离物的连续稀释度（7.0-2.0 log 拷贝/mL），检测到 98.7%（74/75）的鼻咽（NP）拭子稀释度、100%（75/75）的血浆稀释度和 85.3%（64/75）的尿液稀释度。添加核酸稳定剂可显著提高尿液中 MeV RNA 的检测。在每个标本基质中，建立了<3.0 log 拷贝/mL 的 95%下限检测（lower limit of detection，LLOD）。在特异性研究中，未发现与相关病毒或干扰物质的交叉反应。该 MeV 三重靶标 rRT-PCR 检测到了一名确诊麻疹感染个体的临床 NP 拭子中所有三个基因靶标。此外，对 798 例流感 A/B/RSV 阴性儿科 NP 拭子的合并检测发现了两例 MeV RNA 阳性标本，经 N 基因测序证实代表疫苗型麻疹病毒的脱落。
MeV 三重靶标 rRT-PCR 检测方法在三种临床相关基质中对循环 MeV 基因型具有高分析灵敏度。在临床实验室中实施该检测方法可能有助于及时诊断急性麻疹感染并实施适当的感染控制干预措施。
