Jpn. J. Infect. Dis., 68, 455–460, 2015

Original Article Detecting Dengue Nonstructural Protein 1 (NS1) in Urine Samples Using ELISA for the Diagnosis of Dengue Virus

Yuka Saito1,2,MengLingMoi1*, Akira Kotaki1, Makiko Ikeda1, Shigeru Tajima1, Hajime Shiba2, Kuniaki Hosono2, Masayuki Saijo1, Ichiro Kurane3, and Tomohiko Takasaki1 1Department of Virology 1, National Institute of Infectious Diseases, Tokyo 162-8640; 2College of Bioresource Science, Nihon University, Kanagawa 252-0880; and 3National Institute of Infectious Diseases, Tokyo 162-8640, Japan

SUMMARY: Dengue virus (DENV) infection is a serious global health threat. For the surveillance and control of dengue, there is a need for robust diagnostic tools that are relatively easy to use and reliable in various clinical settings. We investigated the applicability of NS1 antigen detection in urine samples for the diagnosis of DENV. About 118 urine samples, obtained from 96 dengue patients at various phases of disease, were used for this study. NS1 antigen was detected by ELISA in the urine samples obtained from patients after 2–17 days of disease onset. Positive detection rates of NS1 antigen ranged between 13–43z. Based on real-time RT-PCR, positive detection rates of viral genome in the urine samples ranged between 20–33z on days 0 to 15. On days 11 to 15 after the disease onset, NS1 antigen was detected at similar rates in serum and urine samples. Additionally, NS1 antigen was detected in 2 urine samples, but not in the serum samples, on days 7 and 16 after the onset of the disease. The results con- firm the applicability of NS1 antigen detection in urine samples using ELISA to diagnose acute DENV infection and suggests that the assay is potentially useful when only limited amounts of serum samples are available and in limited resource settings.

and dengue shock syndrome (10). Currently, there are INTRODUCTION no suitable vaccines available for dengue and also, there Dengue virus (DENV) belongs to the genus Flavi- is a lack of specific treatment. Therefore, early diag- virus, which consists of 4 serotypes (DENV-1, DENV-2, nosis is important for clinical management, both for DENV-3, and DENV-4) (1). DENV infection occurs in prevention of severe disease and infection control. tropical and subtropical areas (2), where it is transmit- Laboratory confirmation of dengue infection includes ted by vector mosquitoes Aedes aegypti and Aedes al- virus isolation, detection of viral genome, viral antigen, bopictus. In recent years, there has been an exponential and a 4-fold rise in antibody titers (10–13). increase in the number of dengue cases worldwide. It NS1 antigen is a useful marker for the laboratory has been speculated that such increase is associated with diagnosis of DENV infection. The NS1 antigen, which increased mosquito activity and range expansion, which is essential for viral replication, is expressed in the intra- have occurred concurrently with global warming and ur- cellular organelles and the cell surface, and also secreted banization (3–5). About 40z of the world's population into the extracellular environment during DENV infec- lives in areas where there is risk of DENV infection tion (14–16). NS1 is detectable in the sera of dengue (5,6). Although DENV has previously not been endemic patients, although the amount of secreted NS1 varies in Japan, the number of imported cases has increased among different individuals and is speculated to be cor- from 32 cases in 2003 to 249 cases in 2013. A recent au- related with the levels of viremia and disease severity tochthonous dengue outbreak in Japan resulted in 160 (17). NS1 antigen is detected in the serum samples after cases (7,8). It is estimated that annually there are 390 the onset of the disease up to 14 days post-infection million cases of dengue infection (9). Although most and after the disappearance of viral genome or in pres- cases of DENV infection are asymptomatic (3), approxi- ence of IgM antibody (13,18). mately 500,000 patients per year are hospitalized as a Serum samples have been proven useful for the result of severe dengue with a mortality rate of 2.5z laboratory diagnosis of DENV infection. In the field, (6,9). In symptomatic infection, dengue infection causes however, or in DHF patients with severe bleeding ten- symptoms that range from mild to severe dency and infants, the use of samples such as urine and life-threatening dengue hemorrhagic fever (DHF) (which can be obtained by non-invasive procedures) is advantageous (19). Previous studies by other investiga- tors have shown the presence of viral genome in the Received October 6, 2014. Accepted December 24, 2014. urine samples of dengue patients (12,20). Additionally, J-STAGE Advance Publication March 13, 2015. NS1 antigen has been detected in urine samples from DOI: 10.7883/yoken.JJID.2014.441 days 2–10 after the onset of the disease (21). However, *Corresponding author: Mailing address: Institute of Trop- determining the applicability of NS1 ELISA at each ical Medicine, Nagasaki University, Sakamoto 1-12-4, phase of the disease would confirm the usefulness of Nagasaki 852-8523, Japan. Tel: ＋81-95-819-7829, Fax: ＋ NS1 antigen detection in urine samples for DENV diag- 81-95-819-7830, E-mail: sherry＠nagasaki-u.ac. jp nosis. In this study, we investigated the use of NS1 Ag

455 ELISA (for urine samples) for the laboratory diagnosis control, positive control, calibrator, and the serum or of DENV infection. NS1 antigen detection rates were urine samples were added to each anti-NS1 monoclonal determined using urine samples obtained from 96 den- antibody-coated microwell. One hundred microliters gue patients in the early and late phases of the disease of diluted conjugate (1×) was then added to each (days 0 to 15). The applicability of NS1 antigen detec- microwell. The samples were mixed in the wells and tion by ELISA using urine samples was also compared incubated for 90 min at 379C. After washing 6 times in to the detection rates of viral genome using real-time 1 × wash buffer, 160 ml of tetramethylbenzidine was RT-PCR and anti-DENV IgM/IgG antibody ELISA. added to the wells and incubated for 30 min at room temperature in the dark. The reaction was terminated with 100 ml stop solution (1N H SO ). Optical density MATERIALS AND METHODS 2 4 (OD) was measured at a wavelength of 450 nm using a Patient serum and urine samples: The panel consisted spectrophotometer. All experiments were performed in of 175 serum samples and 118 urine samples from 96 duplicates. Index or sample ratio was calculated using dengue fever patients who returned to Japan from over- the following formula: mean OD of urine or serum seas travel during 2008–2012. All the samples were sent samples/ mean OD of cut-off controls. Index of ＜0.5, from clinics and hospitals to the National Institute of 0.5–1.0, and ＞1.0 were classified as negative, equivo- Infectious Diseases, Japan, for the diagnosis of arbo- cal, and positive, respectively. Equivocal results were re- virus infection. Patient samples were examined for the garded as negative. presence of the viral genome by fluorogenic real-time Statistical analysis: The tabulation, management, and reverse transcriptase polymerase chain reaction (Taq- analysis of data were performed using Microsoft Excel. Man real-time RT-PCR), and ELISA with NS1 antigen Positive detection rate (z) was calculated using the fol- and anti-DENV antibody (IgM and IgG antibody) lowing formula: the number of positive samples/ the (13,22). For the concentration of urine samples, the total number of samples ×100. The results were com- urine samples were transferred to Vivaspin 20 (Sartorius pared using the chi-squared test and Microsoft Excel. Stedim Biotech, Goettingen, Germany) and centrifuged for 30 min at approximately 1,400 × g. The concen- RESULTS trated urine samples were used at concentration of 10- times that in the original. The day of disease onset was NS1 antigen detection in the serum and urine samples defined as day 0. DENV infection was confirmed by the of DENV patients: We examined the viral genome, NS1 presence of the viral genome and NS1 antigen or a 4- antigen, IgM, and IgG antibody levels in 175 serum fold rise in antibody titers (10). Ten urine samples from samples and 118 urine samples collected from 96 dengue non-dengue patients were used as negative controls. patients. Of the 96 patients, 19 patients were infected Patients were de-identified prior to the conduction of with DENV-1, 17 with DENV-2, 17 with DENV-3, and laboratory tests. The study protocol was approved by 10 with DENV-4. DENV serotype could not be deter- the ethics institutional review board of the National In- minedin33patients. stitute of Infectious Diseases. Positive rates of NS1 antigen were compared between TaqMan real-time RT-PCR: The viral genome in se- the serum and urine samples from days 0 to 15 (Fig. 1) rum and urine samples was detected using real-time RT- after disease onset. Urine samples from non-dengue PCR (20). Viral RNA was extracted from 200 mlofse- fever patients were confirmed to be negative (10/10, rum or 200 ml of urine samples using a High Pure Viral 100z) for the presence of NS1 by ELISA and were used RNA kit (Roche, Madison, WI,USA). For real-time as the negative controls. The filtration method has RT-PCR, 5 mlofRNAwasmixedwithserotype-specific previously proven useful in sample concentration and primer and probe, to make up a final volume of 20 mlof adenovirus detection (23). Therefore, this method was reaction mixture (TaqMan Fast Virus 1-Step Master adapted to concentrate urine samples for dengue virus Mix kit, Life Technologies, Carlsbad, CA, USA) (20). detection in this study. Serum samples from dengue Real-time RT-PCR was used with the following cycle patients were divided into 4 groups according to days conditions: (1) Real-time RT-PCR for 5 min at 509C, after disease onset: 0–5 days, 6–10 days, 11–14 days, followed by 40 cycles of 959C for 3 s and 609Cfor30s. and on and after 15 days. The NS1 antigen positive rate Data was collected and analyzed after 40 cycles. in the serum samples was ＞90z on days 0–10, but Anti-DENV IgM and IgG Antibody: Dengue virus- decreased thereafter to 43z (6/14) on days 11–14 and specific IgM antibodies in serum and urine samples were 12z (3/26) on and after 15 days. In comparison, the determined using IgM capture ELISA (Dengue fever vi- NS1 antigen detection rate in the urine samples was 36z rus IgM capture ELISA, Focus Diagnosis, Cypress, CA, (18/50) on days 0–5, 43z on days 6–10 (19/44), 33z USA) according to manufacturer's instructions. Dengue (3/9) on days 11–14, and 13z (2/15) on and after 15 IgG ELISA (Dengue IgG Indirect ELISA, Panbio, days. The NS1 antigen ELISA index ranged from 2.2 to Queensland, Australia, and Dengue ELISA IgG, Vir- 14.6 in the urine samples determined as positive. cell, Granada, Spain) was used for the detection of anti- Although the NS1 detection rates were higher in the se- DENV IgG antibody according to manufacturer's in- rum samples than in the urine samples during the early structions (13). phase of the disease (days 0–10) (Fig. 1), the NS1 anti- DENV NS1 Antigen ELISA: Dengue NS1 antigen gen was detected in 2 urine samples obtained on day 7 was detected in each sample using a Platelia Dengue and day 16, but not in the serum samples collected on NS1 Ag kit (Bio Rad, Marnes La Coquette, France): the the same days (Table 1). These results indicate that the assay was performed according to the manufacturer's NS1 antigen can be detected in urine samples even after instructions. Briefly, 50 ml of sample diluent, negative NS1 antigen clearance (or decrease to undetectable

456456 Detection of DENV NS1 Antigen in Urine Samples

Fig. 1. Positive rates of (A) NS1 antigen, (B) viral RNA, (C) anti-DENV IgM, and (D) anti-DENV IgG of serum and urine by real-time RT-PCR and ELISA. The number of serum samples in Fig. 1A, 1B, 1C, and 1D were 170 (78 on days 0–5, 52 on days 6–10, 14 on days 11–14, and 26 on 15 days), 118 (71 on days 0–5, 33 on days 6–10, 8 on days 11–14, and 6 on 15 days), 167 (77 on days 0–5, 52 on days 6–10, 14 on days 11–14, and 24 on 15 days), and 170 (78 on days 0–5, 52 on days 6–10, 14 on days 11–14, and 26 on 15 days), respectively. The number of urine samples indicated in Fig. 1A, 1B, 1C, and 1D were 118 (50 on days 0–5, 44 on days 6–10, 9 on days 11–14, and 15 on 15 days). Open bars indicate detection rates using serum samples and closed bars indicate detection rates using urine samples. Asterisk (*) indicates not detected.

Table1.DetectionofNS1antigeninserumandurinebyNS1Ag ELISA

Urine sample1) Total ＋2) －2)

＋ 38 (34z)50(45z)88 Serum samples － 2(2z)21(19z)23 Total 40 71 111

1): Number of positive samples as determined by NS1 Ag ELISA using concentrated urine and non-concentrated urine samples. 2): Number of positive (＋)ornegative(－) samples as determined by NS1 Ag ELISA. Fig. 2. Levels of NS1 antigen in patient #14 in serum, concen- trated urine and non-concentrated urine samples as determined Table 2. Detection of NS1 antigen in concentrated urine and by NS1 Ag ELISA. Paired serum samples, concentrated and non-concentrated urine by NS1 Ag ELISA non-concentrated urine samples were obtained on days 4, 5, 6, Concentrated urine sample 7, 8, 9, 10, and 16 after onset of disease. Open bars indicate de- Total tection rates in serum samples, closed bars indicate detection ＋1) －1) rates in non-concentrated urine samples, and gray bars indicate detection rates in concentrated urine samples. Non-concentrated ＋ 13 (35z)1(3z)14 urine samples － 3(8z)20(54z)23 Total 16 21 37 was detected in 3 concentrated urine samples that were 1): Number of positive (＋)ornegative(－) samples as determined negative before concentration, the results suggest that by NS1 Ag ELISA. the utility of concentrating urine samples for increasing the sensitivity of NS1 antigen ELISA was limited. The NS1 antigen detection rates in 1 dengue patient levels)fromtheserumofsomedenguepatients. (Patient #14) were determined during the course of the Detection of the NS1 antigen was compared between disease. Serum samples obtained on days 4–10 and on 37 paired concentrated urine and non-concentrated day 16 and urine samples obtained on days 5–10 and on urine samples (Table 2). There was no significant differ- day 16 were used (Fig. 2). On days 5–10, all serum and ence in the NS1 antigen detection rates between the con- urine samples tested positive for the NS1 antigen. The centrated urine and non-concentrated urine samples NS1 antigen index values of serum samples, however, (chi-squared test, P ＝ 0.64). Although the NS1 antigen decreased from day 6 onwards (index value ＝15 on

457 days 6–9, index value ＝ 5.9 on day 10). Although the day of disease onset (disease onset day 0), with IgM NS1 antigen index values of the urine samples on days levels increasing from day 3 (13–15,25). The sensitivity 5–10 were lower than those of the serum samples, the and specificity of NS1 Ag ELISA were high, in serum NS1 antigen index values of the urine samples were samples, which demonstrate the utility of the assay in constant on days 6–10 (index value range ＝ 3.2–4.6). dengue diagnosis (13–15). In this study, we evaluated Interestingly, on day 16 after disease onset, the NS1 the utility of NS1 antigen detection in urine samples for antigen was not detected in the patient's serum, but was the laboratory diagnosis of dengue. Real-time RT-PCR detected in the urine. and IgM/IgG antibodies by ELISA compared detection Positive detection rates of the viral genome, NS1 anti- rates of NS1 ELISA in urine samples to those of serum gen, anti-DENV IgM, and anti-DENV IgG: Positive de- samples and detection rates of virus genome. tection rates of the NS1 antigen by NS1 antigen ELISA Serum samples have been routinely used for the were compared to those of the viral genome, anti- laboratory diagnosis of DENV infection. collec- DENV IgM, and anti-DENV IgG in both the serum and tion, however, can be a challenge in areas with limited the urine samples (Fig. 1). The positive detection rate resources and for patients with severe bleeding tendency for the viral genome in the serum samples was 81z andinfants.Insuchcases,urinesamplecollectionis (57/70) on days 0–5, but decreased to 36z (12/33) on more practical (19), as it is non-invasive, rapid, and can days 6–10 (Fig. 1B). In contrast, the viral genome detec- be performed without laboratory instruments such as a tion rates in the urine samples remained at similar levels centrifuge. In this study, we determined the utility of throughout the study period―32z (16/50), 30z NS1 antigen ELISA using urine samples from dengue (13/44), and 33z (3/9) on days 0–5, 6–10, and 11–14, patients. The NS1 antigen was detected in the urine sam- respectively. Viral genome detection rates in the urine ples of dengue patients on days 2–17 after disease onset. samples were higher than those in the serum samples on In a previous study, the NS1 antigen was detected in and after day 11. The NS1 antigen detection rates in the urine samples collected on days 2–10 after disease onset, serum samples were constant on days 6–10 at 90z positive rates of NS1 antigen using NS1 antigen ELISA (47/52), but subsequently decreased to 43z (6/14) on were 68.4z and 63.9z in 19 DF and 36 DHF samples, days 11–14 (Fig. 1A). In contrast, the NS1 antigen de- respectively (21). However, the utility of NS1 antigen tection rates in the urine samples were constant at 36z ELISA was not determined at each disease phase. In the (18/50), 43z (19/44), and 33z (3/9) on days 0–5, 6–10, present study, we determined the detection rates of NS1 and 11–14, respectively. In the serum samples obtained antigen in urine samples at days 0 to 15 after disease on days 6–10, the detection rate of IgM was high at 98z onset and compared the detection rate of NS1 antigen (51/52). The detection rate of the viral genome in the ELISA with that of viral genome (real-time RT-PCR) serum samples was 36z (12/33) (Fig. 1B). On and after and IgM/IgG antibodies. IgM and IgG antibodies were 11 days, the positive rates of IgM antibody detection in detected in 1 urine sample each (1/118, 1z). The results the serum samples increased to 100z (14/14), and the suggest that the detection of IgM and IgG antibody in positive rates of NS1 antigen detection decreased to urine samples is not a feasible method for dengue diag- 43z (6/14) (Fig. 1A and 1C). The IgM antibody detec- nosis. In contrast, NS1 antigen positive rates in urine tion rate in the serum samples was 100z (38/38) on and samples were 36z (18/50) on days 0–5. The rates were after 11 days (Fig. 1C), but the antibody was detected in the highest (43z) on days 6–10 (19/44), and they only 1 urine sample obtained on day 5 (1z, 1/118). decreasedto33z (3/9) on days 11–14 and to 13z Similarly, the IgG antibody detection rate in serum sam- (2/15) on ≧day 15. Viral genome detection rates in ples was 95z (38/40) on and after11 days (Fig. 1D), but urine samples were similar to those of NS1 antigen the IgG antibody was detected in only 1 urine sample ELISA: 32z (16/50) on days 0–5, 30z (13/44) on days obtained on day 12 (1z, 1/118). Although, the viral 6–10, 33z on days 11–14 (3/9), and 20z (3/15) on genome and NS1 antigen were detected in urine samples ≧day 15. We found that NS1 antigen-positive rates using real-time RT-PCR and NS1 antigen ELISA (Fig. were constant (approximately 30z) in urine samples, 1A and 1B), the detection rates in the urine samples although these were lower than those of serum samples were lower than in those in the serum samples (chi- on days 0–10. Of note, in the present study, NS1 antigen squared test: viral genome P ＜ 0.01, NS1 antigen P ＜ was also detected in urine samples at days ＞10 and after 0.01). Concurrent with the results from previous studies the viral genome became undetectable in urine samples. (12), the viral genome was detected in some cases in the Concurrent with the results of previous investigations, urine samples (9/86, 10z), but not in the serum DENV genome was detected at a higher rate in urine samples. The results indicate that the viral genome and samples than in serum samples at days 11 to 15 after dis- antigen are present in urine samples. ease onset (12,26). Although the NS1 antigen detection rates in serum samples were higher than those of urine samples during the early phase of the disease, during the DISCUSSION late phase of the disease (beyond day 11), NS1 antigen Dengue, a global public health problem, is also a seri- was constantly detected at similar rates in urine and se- ous health threat in areas with limited resources and rum samples. The results suggest that detection of NS1 access to diagnostic tools (9,10,24). For dengue disease antigen in urine samples, using NS1 ELISA, is particu- surveillance and control, there is a need for robust diag- larly useful at the late phase of the disease. Interesting- nostic tools that are relatively easy to use and reliable in ly, on days 11 to 15 after disease onset, NS1 antigen de- various clinical settings. NS1 antigen ELISA has tection rates in urine and serum samples were at similar emerged as a promising diagnostic tool and is relatively levels. It has been hypothesized that DENV infection easy to use (14). NS1 antigen could be detected from the deposition of virus-antibody immune-complexes in vas-

458458 Detection of DENV NS1 Antigen in Urine Samples cular and glomerular tissue (27–29). There remains a sion by mosquitoes. Curr Trop Med Rep. 2014;1:21-31. possibility that the prolonged detection of virus genome 7. Ministry of Labour, Health and Welfare of Japan. Autochtho- nous Dengue cases in Japan (Report no. 16). Available at . Accessed on September 22, 2014. Japanese. Overall, the results suggest the utility of urine samples 8.KutsunaS,KatoY,MoiML,etal.Autochthonous dengue fever, obtained up to day 17 after disease onset for laboratory Tokyo, Japan, 2014. Emerg Infect Dis. 2015;21:517-20. diagnosis for DENV infection by NS1 antigen ELISA. 9. Bhatt S, Gething PW, Brady OJ, et al. The global distribution and burden of dengue. Nature. 2013;496:504-7. The filtration method has been previously applied to 10. World Health Organization. Dengue. Guidelines for diagnosis, concentrate virus (23). In the present study, there was treatment, prevention and control. Available at . concentrated and non-concentrated urine samples (chi- Accessed on September 22, 2014. squared test, P ＝ 0.64). Interestingly, NS1 antigen was 11. Hang VT, Nguyet NM, Trung DT, et al. Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, detected in serum samples of DENV-4 patients, but not specificity and relationship to viraemia and antibody responses. in urine samples. Using the present ELISA method, PLoS Negl Trop Dis. 2009;3:e360. other investigators have demonstrated that the NS1 12. Hirayama T, Mizuno Y, Takeshita N, et al. Detection of dengue antigen-positive rate of serum samples from DENV-4 virus genome in urine by real-time reverse transcriptase PCR: a laboratory diagnostic method useful after disappearance of the patients (19z) was lower than that of other DENV sero- genome in serum. J Clin Microbiol. 2012;50:2047-52. types (DENV1, 67z; DENV-2, 68z; DENV-3, 69z) 13. Moi ML, Omatsu T, Tajima S, et al. Detection of dengue virus (30). Further studies using a higher number of urine nonstructural protein 1 (NS1) by using ELISA as a useful labora- samples obtained from DENV-4 patients are expected to tory diagnostic method for dengue virus infection of international address the discrepancies in NS1 antigen detection rates travelers. J Travel Med. 2013;20:185-93. 14. Alcon S, Talarmin A, Debruyne M, et al. Enzyme-linked im- between serotypes. Overall, the results suggest that munosorbent assay specific to Dengue virus type 1 nonstructural where serum sample collections are not practical, urine protein NS1 reveals circulation of the antigen in the blood during samples may be a feasible alternative. the acute phase of disease in patients experiencing primary or sec- The utility of urine samples for laboratory diagnosis ondary . J Clin Microbiol. 2002;40:376-81. 15. Kumarasamy V, Chua SK, Hassan Z, et al. 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Kinetics of antibodies in Acknowledgments Wewouldliketothankthehealthcareprac- sera, saliva, and urine samples from adult patients with primary titioners of the clinics and hospitals in Japan for providing us with the or secondary dengue 3 virus infections. Int J Infect Dis. 2007;11: serum and urine samples for laboratory diagnosis of dengue. This 256-62. work was supported by funding from the Research on Emerging and 20. Mizuno Y, Kotaki A, Harada F, et al. Confirmation of dengue vi- Re-emerging Infectious Diseases by the Ministry of Health, Labour rus infection by detection of dengue virus type 1 genome in urine and Welfare, Japan (grants H23-shinkou-ippan-010 and H26- and saliva but not in plasma. Trans R Soc Trop Med Hyg. 2007; shinkou-jitsuyouka-007) and a grant-in-aid for Scientific Research 101:738-9. (Wakate B no. 26870872) from the Ministry of Education, Culture, 21. Chuansumrit A, Chaiyaratana W, Tangnararatchakit K, et al. Sports, Science and Technology of Japan. 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