Please cite this article in press as: Korhonen EM, et al. Approach to non-invasive sampling in dengue diagnostics: Exploring virus and NS1 antigen detection in saliva and urine of travelers with dengue. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.08.021 ARTICLE IN PRESS G Model JCV 3116 1–6 Journal of Clinical Virology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Clinical Virology jou rn al hom epage: www.elsevier.com/locate/jcv Approach to non-invasive sampling in dengue diagnostics: Exploring virus and NS1 antigen detection in saliva and urine of travelers with dengue Essi M. Korhonen a,∗ , Eili Huhtamo a , Anna-Maija K. Virtala b , Anu Kantele c,d,1 , Q1 Olli Vapalahti a,b,e,1 a Department of Virology, Haartman Institute, Faculty of Medicine, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland b Division of Microbiology and Epidemiology, Department of Basic Veterinary Sciences, P.O. Box 66 (Agnes Sjöbergin katu 2), FIN-00014 University of Helsinki, Finland c Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland d Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland e Department of Virology and Immunology, Helsinki University Central Hospital Laboratory (HUSLAB), P.O. Box 400 (Haartmaninkatu 3), 00029 HUS, Finland a r t i c l e i n f o Article history: Received 24 June 2014 Received in revised form 22 August 2014 Accepted 24 August 2014 Keywords: Dengue diagnostics NS1 RNA Real-time RT-PCR Urine Saliva Non-invasive a b s t r a c t Background: Dengue diagnostics currently relies on serum and plasma tests. Although the proof of concept for detecting dengue virus (DENV) RNA and nonstructural protein 1 (NS1) antigen from urine and saliva has been demonstrated, few studies have explored their use in diagnostics. Objectives: To investigate the occurrence, excretion kinetics, and diagnostic potential of DENV-RNA and NS1 antigen in the urine and saliva of dengue patients. Study design: We examined serial serum, urine (n = 50) and saliva (n = 48) samples of 14 Finnish travelers with dengue. All samples were analyzed by NS1 ELISA and DENV RT-PCR, and the first and last serum specimens were tested for DENV IgG and IgM. In addition, biochemical parameters were studied from the urine and clinical and laboratory data of the patients were collected. Results: DENV-NS1 protein and RNA proved detectable from saliva and urine using tests developed for serum samples. RNA/NS1 detection showed a diagnostic sensitivity of 64%/54% and 60%/56% for urine and saliva, respectively. RNA analyses performed on days 7–13 after onset of symptoms revealed the sensitivity for urine (72%) to be greater than for serum (31%) or saliva (50%). The concentration of urine samples had no impact on RNA detection. Conclusions: Noninvasive sampling enables an alternative approach to dengue diagnostics. The perfor- mance of the NS1 antigen assay may be improved by optimizing it for urine and saliva samples. The prolonged excretion of DENV-RNA in urine extends the sampling time window for molecular diagnostics and surveillance. © 2014 Published by Elsevier B.V. 1. Background With worldwide distribution in the tropics and subtropics, dengue accounts for the majority of mosquito-borne viral diseases, totaling annually 100 million clinical infections [1,2]. The manifes- tations of DENV infection vary from asymptomatic to mild flu-like ∗ Corresponding author. Tel.: +358 50 5448282; fax: +358 91 9126491. E-mail addresses: essi.m.korhonen@helsinki.fi, essi.korhonen@redcross.fi (E.M. Korhonen). 1 Shared last authorship. disease, and further to potentially lethal severe dengue manifes- ting as hemorrhagic complications or shock [2]. Early diagnosis is valuable not only for patient management but also for control- ling outbreaks and preventing the spread of DENV to non-endemic areas. The current diagnostics relies on detecting dengue-specific antibodies (IgG and IgM), DENV-NS1 protein, DENV-RNA, and, occasionally, infectious virus from serum or plasma. In the early diagnostics, testing for NS1 antigen or viral genome are the meth- ods of choice, since the antibody response is, particularly in primary infections, generated only several days after onset of symptoms. As antibodies to other flaviviruses are cross-reactive with DENV, http://dx.doi.org/10.1016/j.jcv.2014.08.021 1386-6532/© 2014 Published by Elsevier B.V. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46