1. Viral RNA Degradation Makes Urine a Challenging Specimen for Detection of Japanese Encephalitis Virus in Patients With Suspected CNS Infection
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Bharucha, Tehmina, Sengvilaipaseuth, Onanong, Seephonelee, Malee, Vongsouvath, Malavanh, Vongsouvath, Manivanh, Rattanavong, Sayaphet, Piorkowski, Géraldine, Lecuit, Marc, Gorman, Christopher, Pommier, Jean-David, Garson, Jeremy, Newton, Paul, De Lamballerie, Xavier, Dubot Pérès, Audrey, Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Mahidol University [Bangkok]-Mahosot Hospital, University College of London [London] (UCL), Unité des Virus Emergents (UVE), Aix Marseille Université (AMU)-Institut de Recherche pour le Développement (IRD)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service des Maladies infectieuses et tropicales [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Biologie des Infections - Biology of Infection, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Nuffield Department of Clinical Medicine [Oxford], University of Oxford [Oxford], The research has been implemented in collaboration with the ComAcross project (www.onehealthsea.org/comacross) thanks to the financial support of the European Union (EuropeAid, INNOVATE contract 315-047). It was also supported by Total Foundation, Horizon 2020 research and the innovation program EVAg under grant agreement No. 653316, the Institute of Research for Development, Aix-Marseille University, and the Wellcome Trust of Great Britain., We are very grateful to the patients and to Asst. Prof. Bounthaphany Bounxouei, the Director of Mahosot Hospital, the late Dr. Rattanaphone Phetsouvanh, Director of the Microbiology Laboratory, Dr. Mayfong Mayxay, and the staff of the wards and Microbiology Laboratory of Mahosot Hospital. We also thank Ooyanong Phonemixay for technical help and support, Asst. Prof. Dr. Bounnak Saysanasongkham, Director of the Department of Health and Rehabilitation (DHR), Ministry of Health, and Prof. Bounkong Syhavong, Minister of Health, Lao P.D.R., for their very kind help and support. We thank the stakeholders of the ongoing SEAe project, Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and University of Oxford
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Japanese encephalitis virus ,diagnosis ,viruses ,RT-qPCR ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,flavivirus ,RNA - Abstract
International audience; Background: 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.Methods: 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.Results: 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.Conclusions: 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.
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- 2019