Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome
Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome
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DOI：
10.1128/jcm.01902-06
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发表时间：
2007-08-01
影响因子：
9.4
通讯作者：
Allard, Annika
中科院分区：
文献类型：
--
作者：
Evander, Magnus;Eriksson, Irene;Allard, Annika
普马拉病毒 (PUUV) 是瑞典北部的地方性汉坦病毒，可引起流行性肾病 (NE)，这是一种较轻的肾综合征出血热。需要快速、可靠的诊断来区分该疾病与其他感染。通过比对从 11 只不同银行田鼠和一名人类患者中分离出的病毒 RNA 序列，我们设计了一种用于检测 PUUV RNA 的实时逆转录酶 (RT) PCR 方法。实时 RT-PCR 检测显示 20 至 2 X 106 个病毒拷贝呈线性，所有实验的相关系数均高于 0.98 至 0.99。 PUUV cDNA 的检测阈值是每个反应两个拷贝。用于检测 PUUV RNA 的两步定性 RT-PCR 与实时 RT-PCR 检测结果显示 100% 一致。流行地区的 34 名患有典型临床 NE 疾病的 PUUV 免疫球蛋白 M (IgM) 阳性患者中，有 33 名检测到 PUUV RNA 病毒血症。一份 PUUV IgM 阴性样本含有 PUUV RNA，4 天后，该患者的 IgM 呈阳性。在 IgM 不确定的样本中，43% 呈 PUUV RNA 阳性。研究了抗体滴度和 PUUV 病毒血症的动力学，6 名 NE 患者中有 5 名在疾病爆发几天后表现出 PUUV 病毒血症减少，同时 PUUV IgM 和 IgG 增加。对于一名 PUUV RNA 水平持续较高但 IgM 较低且无 IgG 反应的患者，感染是致命的。这些发现表明，实时 RT-PCR 是诊断 PUUV 病毒血症和在 IgM 抗体出现之前的早期时间点检测 PUUV RNA 的有用方法。
Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 X 106 virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.