Bharucha Tehmina, Sengvilaipaseuth Onanong, Seephonelee Malee, Vongsouvath Malavanh, Vongsouvath Manivanh, Rattanavong Sayaphet, Piorkowski Géraldine, Lecuit Marc, Gorman Christopher, Pommier Jean-David, Garson Jeremy A, Newton Paul N, de Lamballerie Xavier, Dubot-Pérès Audrey
Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao P.D.R.
Division of Infection and Immunity, University College London, London, UK.
Open Forum Infect Dis. 2019 Feb 6;6(3):ofz048. doi: 10.1093/ofid/ofz048. eCollection 2019 Mar.
Japanese encephalitis virus (JEV) is a leading cause of central nervous system (CNS) infections in Asia and results in significant morbidity and mortality. JEV RNA is rarely detected in serum or cerebrospinal fluid (CSF), and diagnosis of JEV infection is usually based on serological tests that are frequently difficult to interpret. Unlike serum or CSF, urine is relatively easy to obtain, but, to date, there has been minimal work on the feasibility of testing urine for JEV RNA.
We investigated the use of lysis buffer and a Microsep device to optimize urine storage for detection of JEV RNA by reverse transcription real-time polymerase chain reaction (RT-qPCR). The best of the studied methods was then evaluated in consecutive patients admitted to the hospital with suspected CNS infections in Laos.
We demonstrated degradation of JEV RNA in urine after even short storage periods at 4°C or -80°C. Although there was no advantage in using a Microsep concentration device alone, immediate addition of lysis buffer to fresh urine improved the detection of JEV RNA at the limit of detection.
In 2 studies of 41 patients with acute encephalitis syndrome, 11 (27%) were positive for JEV IgM in CSF and/or serum, and 2 (4.9%) were JEV RT-qPCR positive from throat swabs. JEV RNA was not detected in any of these patients' urine samples. However, lysis buffer was only used during a prospective study, that is, for only 17/41 (41%) patient urine samples. Our findings suggest a need for larger studies testing urine for JEV RNA, with urine collected at different times from symptom onset, and using lysis buffer, which stabilizes RNA, for storage.
日本脑炎病毒（JEV）是亚洲中枢神经系统（CNS）感染的主要病因，可导致显著的发病率和死亡率。在血清或脑脊液（CSF）中很少检测到JEV RNA，JEV感染的诊断通常基于血清学检测，而这些检测结果往往难以解读。与血清或脑脊液不同，尿液相对容易获取，但迄今为止，关于检测尿液中JEV RNA可行性的研究极少。
我们研究了使用裂解缓冲液和Microsep装置来优化尿液储存条件，以便通过逆转录实时聚合酶链反应（RT-qPCR）检测JEV RNA。然后，在老挝一家医院连续收治的疑似CNS感染患者中评估了所研究的最佳方法。
我们发现，即使在4°C或-80°C下短期储存后，尿液中的JEV RNA也会降解。虽然单独使用Microsep浓缩装置没有优势，但在新鲜尿液中立即添加裂解缓冲液可提高在检测限水平对JEV RNA的检测。
在对41例急性脑炎综合征患者的两项研究中，11例（27%）脑脊液和/或血清中的JEV IgM呈阳性，2例（4.9%）咽喉拭子的JEV RT-qPCR呈阳性。在这些患者的任何尿液样本中均未检测到JEV RNA。然而，裂解缓冲液仅在前瞻性研究中使用，即仅用于17/41（41%）例患者的尿液样本。我们的研究结果表明，需要开展更大规模的研究，在症状出现后的不同时间收集尿液，并使用可稳定RNA的裂解缓冲液进行储存，以检测尿液中的JEV RNA。
