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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

Background

Dengue virus ( DENV) nonstructural protein 1 ( NS1) has been used as a novel diagnostic marker during the early phase of DENV infection.

Methods

Presence of NS1 antigen was examined using 336 serum samples obtained from 276 travelers returning to Japan from Asia, Central and South America, Pacific Islands, and Africa with dengue. Assay specificity was evaluated using 148 non-dengue samples.

Results

Positive rates among four DENV serotypes were 68%–89%. NS1 antigen positive rates were at similar levels between primary infection and secondary infection. NS1 antigen positive rates were 88%–96% on days 1–5, 75%–100% on days 6–10, and 36–60% on ≥day 11. Positive rates using real-time polymerase chain reaction (RT-PCR) were over 70% on days 1–5, but decreased thereafter.

Conclusions

The results indicate that NS1 antigen positive rates were higher than those of RT-PCR during longer period of early phase in DENV infection. Thus, NS1 antigen ELISA is a useful tool for confirming DENV infection in international travelers, when it is used in combination with anti-DENV IgM ELISA.

Dengue virus (DENV) infection is a major health problem in tropical and subtropical regions. The disease is estimated to affect 50 million people annually worldwide.[1] It has been suggested that the spread of dengue epidemics in the present decade has been caused by increased international travel and urbanization.[2-4] Recently, DENV transmission has been documented in previously nonendemic areas, including Nepal, Bhutan, and France.[5-7] The number of imported dengue cases has also increased in nonendemic countries such as Japan, where there was more than a twofold increase in DENV cases from 92 in 2009 to 245 in 2010.[8]

Infection with any of the four DENV serotypes causes a range of symptoms: from mild undifferentiated fever to the more severe and sometimes fatal, dengue hemorrhagic fever and dengue shock syndrome.[9-11] No specific therapeutics are available to treat the disease. Early disease confirmation is essential for clinical management as some patients' symptoms change from mild to severe disease in a short period of time.[12] In nonendemic countries with low public recognition of DENV or in areas where DENV laboratory diagnosis is not routinely performed, laboratory diagnosis is essential for differential diagnosis, outbreak investigation, surveillance, and for the facilitation of public health policy decisions.

Due to the complexity of DENV confirmation, virus isolation, detection of viral genome or a fourfold rise in antibody titers between acute- and convalescent-phase serum samples are required for confirmatory diagnosis.[12] An ideal diagnostic test would be affordable and easy to use with high performance and sensitivity in different health settings. In addition, it would be an advantage if the diagnostic assay were flexible in accommodating various laboratory conditions such as in the retainment of assay sensitivity when only limited amounts of serum sample were available. Recently, commercial ELISA tests that detect the nonstructural protein 1 (NS1) have offered a new platform for DENV diagnosis, and studies have shown that detection of NS1 antigen could be useful for the confirmation of DENV infection.[13, 14] In this study, we examined the utility of NS1 antigen detection in laboratory diagnosis of DENV infection using a panel of serum samples from travelers. The NS1 antigen positive rates determined by NS1 ELISA were compared with the positive rates of real-time polymerase chain reaction (RT-PCR) and IgM-ELISA. The results suggest that NS1 antigen ELISA is useful for confirming DENV infection, particularly when utilizing serum samples obtained 1–10 days after the onset of disease.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

Patient Serum Samples

The serum panel consisted of 336 serum samples from cases confirmed positive for DENV infection by RT-PCR, and anti-DENV IgM and IgG antibody. The serum samples were collected from patients admitted in clinics and hospitals in Japan from the years 2007–2011, and sent to the National Institute of Infectious Diseases, Japan for laboratory diagnosis of dengue. Additionally, the panel included 148 serum samples collected from patients with other illnesses that tested negative for DENV by RT-PCR and serology. The history of Japanese encephalitis and yellow fever vaccination of each traveler was not ascertained. All serum samples were de-identified prior to the conduction of laboratory diagnostic tests. The information of the countries visited was obtained for 276 patients. A total of 191 (69%) returned from Southeast Asia, 56 (20%) from South Asia, 13 (5%) from Central and South America, 11 (4%) from the Pacific Islands, 4 (1%) from Africa, and 1 (0.4%) from the Middle East. Day 1 after onset of disease is defined as the day when the first symptoms such as fever were identified.[15] Primary infection was defined by the positive detection of viral RNA with the absence of DENV anti-DENV IgG antibodies and the absence or presence of anti-DENV IgM antibodies. Secondary infection was defined by the presence of anti-DENV IgG antibodies at the stage of the absence of anti-DENV IgM antibodies.[15] The definition was also supported by DENV E/M protein-specific IgM/IgG OD ratios of <1.2 (secondary infection).[12] The study protocol was approved by the institutional review board of the National Institute of Infectious Diseases, Japan.

RT-PCR

Detection of DENV RNA by RT-PCR was performed as reported previously (Tables 1 and 3).[15, 16] Viral RNA was extracted using High Pure Viral RNA extraction kit (Roche Diagnostics, Mannheim, Germany) and DENV serotypes were determined by serotype-specific RT-PCR.[15, 16]

Anti-DENV IgM and IgG Antibody ELISA

Dengue-virus specific IgM antibody in serum samples was determined using IgM capture ELISA (Dengue Fever Virus IgM Capture ELISA, Focus Diagnostics, CA, USA) according to the manufacturer's instructions. Dengue indirect IgG ELISA (Panbio, Queensland, Australia) was used for the detection of anti-DENV IgG antibody according to the manufacturer's instructions.[15]

Dengue NS1 Antigen ELISA

Detection of the NS1 antigen was performed using Platelia Dengue NS1 Antigen (Biorad Laboratories, Marnes-la-Coquette, France) and Pan-E Dengue Early ELISA (Panbio) according to manufacturers' instructions. The former kit was mainly used in the study. For the Platelia Dengue NS1 Antigen ELISA kit, 50 μL of serum sample, 50 μL of sample diluent (Diluent R7, phosphate buffer, Tween 20, and fetal calf serum supplemented with 0.15% ProClinTM 300) and 100 μL of diluted conjugate (anti-NS1 monoclonal antibody-coated to horseradish peroxidase supplemented with 0.15% ProClinTM 300) were added to each anti-NS1 monoclonal antibody coated well. The assay plate was incubated at 37°C for 90 minutes. Positive controls and negative controls with calibrator sera were included in each assay. After six washings, 160 μL of tetramethylbenidine (TMB) substrate was added to each of the wells and the plate was further incubated at room temperature for 30 minutes in the dark. Reaction was terminated with 100 μL of stop solution (1 N H2SO4). Optical density (OD) readings were obtained with a spectrophotometer at wavelengths of 450 nm/620 nm. The index of each sample was calculated with the following formula: OD of samples/OD of calibrators. As the Biorad NS1 ELISA kit showed high sensitivity using 50 μL of patient serum samples, the serum sample volume was reduced and the assay was tested for detection rates. Serum samples were first diluted to 1:10 or 1:100 using diluent (Diluent R7, Platelia Dengue NS1 Ag, Biorad). The assay was then performed according to manufacturer's instructions (Platelia Dengue NS1 Ag, Biorad). Results were interpreted in accordance with manufacturer's recommendations. Sample ratios were determined by dividing the sample OD with the cut-off OD. Sample ratios of <0.5, 0.5–1.0, and >1.0 were classified as negative, equivocal, and positive, respectively. Equivocals were regarded as negative for analysis.[14, 17]

For the Panbio kit (Panbio Diagnostics, Queensland, Australia), 100 μL of diluted (1:10 in serum diluent) patient serum sample, positive control, negative control, or calibrator was added to microwells pre-coated with anti-NS1 antibody. The plate was incubated at 37°C for 1 hour. After washing six times, the plates were incubated at 37°C following the addition of HRP-conjugated anti-NS1 mAb. The wells were then washed six times and TMB was added to the wells. The plates were further incubated at room temperature for 10 minutes in the dark. The reaction was stopped with stop solution (1 M H3PO4) and the plates were read. The index value was determined by dividing the average OD of each sample by the cut-off value. The cut-off value was determined by multiplying the average OD of the calibrator by the calibration factor provided by the manufacturer. Index values of <0.9, 0.9–1.1, and >1.1 were considered negative, equivocal, and positive, respectively. Equivocals were regarded as negative.[14, 17]

Statistical Analyses

Tabulation, management, and analysis of raw data were performed using Microsoft Excel. Detection rates were determined as reported previously.[18, 19] Study outcomes were compared by chi-squared test and Fisher's exact test (for small sample sizes with n < 10), using the QI Macros statistical analysis program (KnowWare International, Denver, CO, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

Detection of NS1 Antigen in Serum Samples From Confirmed Cases with DENV Infection Using NS1 Antigen Detection ELISA

A total of 484 serum samples collected from 2007 to 2011 were used in the study. In 336 serum samples DENV infection was confirmed; 301 (90%) were found to be NS1 positive using the Biorad assay. All 148 serum samples from confirmed dengue-negative donors yielded negative results. Of 336 samples tested by both the NS1 antigen ELISA and RT-PCR, 223 (66%) were positive by both the NS1 antigen ELISA and RT-PCR. A total of 78 samples (23%) were positive by NS1 antigen ELISA but negative by RT-PCR, 3 (1%) samples were negative by the NS1 antigen ELISA but positive by RT-PCR, and 32 samples were negative by both NS1 ELISA and RT-PCR. Detection rates between NS1 ELISA and RT-PCR were statistically significant (chi-squared test, p < 0.01). Of the 336 serum samples from DENV confirmed patients, 199 serum samples were performed in parallel with the Panbio assay. The Panbio assay identified 102 (51%) as NS1 positive of 199 serum samples from DENV infection confirmed cases. Additionally, the Panbio assay identified all 24 serum samples from confirmed dengue-negative cases as negative. The p value (chi-squared test, p < 0.01) suggests that the levels of sensitivity between the two kits were statistically different.

The rate of detection using RT-PCR was 76% during the early phase (1–5 days after onset of disease) but decreased later during infection (days 6–10 = 20%, ≥11 days = 0%) (Table 1). In contrast, the rate of detection using NS1 was 93% (days 1–5) and 91% (days 6–10). As with RT-PCR, the rate of NS1 detection decreased to 50% at the later phase of infection (≥11 days) (Table 1). The rate of detection using IgM was lower (60%) as compared to RT-PCR and NS1 during the early phase of the disease (days 1–5), but was high during the later phase of disease (days 6–10 = 90% and ≥11 days = 93%, Table 2).

Table 1. Detection rates of NS1 antigen by ELISA and/or viral RNA by RT-PCR
DayaSample numberType of test or combination (% of total)
NS1 resultsNDb++++
  ANDAND OR
RT-PCR results++ND+
  1. a

    Days after onset of disease.

  2. b

    Number of positive (+) or negative (−) as determined by NS1 ELISA or RT-PCR. ND indicates not determined.

  3. c

    Number of samples ( Ntotal = 290) with indicated results/total number of samples (percentage of samples with indicated results, %).

Day 123 23 (100)c22 (96)0 (0)22 (96)23 (100)
Day 224 22 (92)21 (88)0 (0)21 (88)22 (92)
Day 346 33 (72)33 (72)9 (20)42 (91)42 (91)
Day 436 27 (75)27 (75)7 (19)34 (94)34 (94)
Day 541 24 (59)24 (59)15 (37)39 (95)39 (95)
Subtotal Days 1 to 5170 129 (76)127 (75)31 (18)158 (93)160 (94)
Day 629 9 (31)9 (31)17 (59)26 (90)26 (90)
Day 730 5 (17)5 (17)22 (73)27 (90)27 (90)
Day 816 3 (19)3 (19)13 (18)16 (100)16 (100)
Day 98 1 (13)1 (13)5 (63)6 (75)6 (75)
Day 107 0 (0)0 (0)7 (100)7 (100)7 (100)
Subtotal Days 6 to 1090 18 (20)18 (20)64 (71)82 (91)82 (91)
Day 1110 0 (0)0 (0)6 (60)6 (60)6 (60)
Day 127 0 (0)0 (0)4 (57)4 (57)4 (57)
Day 132 0 (0)0 (0)1 (50)1 (50)1 (50)
Day ≥1411 0 (0)0 (0)4 (36)4 (36)4 (36)
Subtotal Days 11 to ≥1430 0 (0)0 (0)50 (15)15 (50)15 (50)
Total290 147 (51)145 (50)145 (50)255 (88)257 (89)
Table 2. Detection rates of NS1 antigen by ELISA and/or anti-DENV IgM antibody by ELISA
DayaSample numberType of test or combination of test (% of total)
NS1 resultsNDb++++
  ANDAND OR
IgM results++ND+
  1. a

    Days after onset of disease.

  2. b

    Number of positive (+) or negative (−) as determined by NS1 ELISA or RT-PCR. ND indicates not determined.

  3. c

    Number of samples ( Ntotal = 290) with indicated results/total number of samples (percentage of samples with indicated results, %).

Day 123 5 (22)c5 (22)17 (74)22 (96)22 (96)
Day 224 5 (21)5 (21)16 (67)21 (88)21 (88)
Day 346 21 (46)21 (46)21 (46)42 (91)42 (91)
Day 436 32 (89)32 (89)2 (6)34 (94)34 (94)
Day 541 39 (95)39 (95)0 (0)39 (95)39 (95)
Subtotal Days 1 to 5170 102 (60)102 (60)56 (33)158 (93)158 (93)
Day 629 26 (90)26 (90)0 (0)26 (90)26 (90)
Day 730 29 (97)27 (90)0 (0)27 (90)29 (97)
Day 816 13 (81)13 (19)3 (19)16 (100)16 (100)
Day 98 7 (88)6 (75)0 (0)6 (75)7 (88)
Day 107 6 (86)6 (86)1 (14)7 (100)7 (100)
Subtotal Days 6 to 1090 81 (90)82 (91)4 (4)82 (91)85 (94)
Day 1110 8 (80)6 60)0 (0)6 (60)8 (80)
Day 127 7 (100)4 (57)0 (0)4 (57)7 (100)
Day 132 2 (100)1 (50)0 (0)1 (50)2 (100)
Day ≥1411 11 (100)4 (36)0 (0)4 (36)11 (100)
Subtotal Days 11 to ≥1430 28 (93)15 (50)0 (0)15 (50)28 (93)
Total290 211 (73)199 (69)60 (21)255 (88)271 (93)

NS1 Antigen Positive Rates and Positive Rates by RT-PCR and IgM ELISA

Positive rates by NS1 antigen ELISA were compared to positive rates by RT-PCR and IgM ELISA from day 1 to day ≥14 (Figure 1). Because there was no difference in NS1 antigen positive rates in primary and secondary DENV infections, the data were combined and analyzed. NS1 antigen positive rates were 88%–96% on days 1–5, 75%–100% on days 6–10, and 36%–60% on ≥day 11 (Figure 1). RT-PCR positive rates were over 70% on days 1–5 (Figure 1); however, positive rates were low or there were no positive samples on days 6–10 and ≥11 days. IgM positive rate was 60% on days 1–5, but were nearly 100% on days 6–10 and ≥11 days.

image

Figure 1. Positive rates of NS1 ELISA and anti-DENV IgM on days 1 to ≥14 after disease onset. (A) Detection of NS1 antigen using the Biorad NS1 Ag ELISA using 290 serum samples. Closed bars indicate percentage of positive detection of viral genome by RT-PCR. Open bars indicate percentage of positive detection of NS1 antigen by NS1 Ag ELISA. Closed circles indicate percentage of positive detection of anti-DENV IgM by ELISA. (*) No samples were positive by RT-PCR.

Download figure to PowerPoint

The rate of detection of each assay alone was 88% for NS1 assay, 73% for IgM ELISA, and 51% for RT-PCR. NS1 Ag ELISA in combination of RT-PCR yielded a detection rate of 89% (chi-squared test, p = 0.80 in comparison to NS1 ELISA alone, Tables 1 and 2). Although the rate of detection using the NS1 ELISA in combination with RT-PCR was 93% from days 1–5 and days 6–10 after onset of disease, the rate of detection was 50% from ≥11 days after onset of disease. The detection rates of NS1 in combination with IgM ELISA (detection rate = 93%, chi-squared, p = 0.02 in comparison to NS1 ELISA) was, however, consistently above 90% at days 1–5, days 6–10, and ≥11 days after onset of disease. Thus, the results suggest that a combination of NS1 ELISA and IgM ELISA was sufficient to yield a 93% detection rate of dengue cases from days 1–5, days 6–10, to ≥11 days in our study (Table 2).

NS1 Antigen Positive Rates Among the Four DENV Serotypes

NS1 antigen positive rates were compared among four DENV serotypes. Positive rates were from 68% to 89% (DENV-1 = 89%; DENV-2 = 82%; DENV-3 = 81%; DENV-4 = 68%) using Biorad NS1 antigen ELISA (Table 3). The detection rate of the NS1 Biorad assay from days 1–10 after onset of disease for DENV-1 was 92/95 (97%), DENV-2 = 53/62 (85%), DENV-3 = 61/71 (86%), and DENV-4 = 26/31 (84%). On day 11 and after, rate of detection of the NS1 for DENV-1 was 31% (4/13), DENV-2 = 40% (2/5), DENV-3 = 16% (1/6), and DENV-4 = 0% (0/7). As the number of serum samples examined in days ≥11 after onset of disease was small, detection rates between serotypes were compared with those on days 1–10 after onset of disease. The detection rate of NS1 was highest using samples from DENV-1 patients (97%) as compared to detection rates of pooled serotypes (85%, Fisher's exact test, p < 0.01, days 1–10). The differences between detection rates of DENV-2, DENV-3, and DENV-4 for days 1–10 were not statistically significant (Fisher's exact test, p > 0.05).

Table 3. NS1 antigen positive rates by NS1 antigen ELISA in the four DENV serotypes
DayaInfecting DENV serotype (%)
DENV-1bDENV-2bDENV-3bDENV-4b
  1. a

    Days after onset of disease.

  2. b

    Infecting serotype as determined by RT-PCR.

  3. c

    Number of samples ( Ntotal = 290) with indicated results/total number of samples (percentage of samples with indicated results, %).

  4. d

    ND indicates that the value could not be determined due to an insufficient number of samples.

115/15 (100)c8/9 (89)13/15 (87)2/2 (100)
211/11 (100)5/7 (71)5/7 (71)3/4 (75)
312/13 (92)15/17 (88)14/16 (88)6/6 (100)
49/10 (90)9/10 (90)8/9 (89)2/3 (67)
516/16 (100)8/8 (100)9/9 (100)6/6 (100)
1 to 563/65 (97)45/51 (88)49/56 (88)19/21 (90)
69/9 (100)4/4 (100)4/4 (100)3/4 (75)
77/8 (88)1/1 (100)3/4 (75)2/3 (67)
83/3 (100)3/4 (75)4/5 (80)1/1 (100)
95/5 (100)0/1 (0)1/2 (50)1/2 (50)
105/5 (100)0/1 (0)0/0 (NDd)0/0 (ND)
6 to 1029/30 (97)8/11 (72)12/15 (80)7/10 (70)
112/2 (100)0/1 (0)1/2 (50)0/1 (0)
120/1 (0)1/1 (100)0/1 (50)0/0 (ND)
131/1 (100)0/0 (ND)0/0 (ND)0/1 (0)
≥141/9 (11)1/3 (33)0/3 (0)0/5 (0)
≥114/13 (31)2/5 (40)1/6 (16)0/7 (0)
Total by serotype96/108 (89)55/67 (82)62/77 (81)26/38 (68)

NS1 Antigen Positive Rates in Primary and Secondary DENV Infections

DENV antigen NS1 positive rates by ELISA were compared in primary and secondary DENV infections from days 1–5, days 6–10, and ≥11 days. Positive rates were at similar levels in primary and secondary DENV infections (Table 4). At days 1–5 after onset of disease, the mean IgG index for secondary infection was 2.1 (positive >1.1) and primary infection serum samples were negative for IgG (mean IgG index for primary infection = 0.7). The rate of detection of NS1 antigen was 94% for secondary infection and 91% for primary infection (chi-squared test, p = 0.54, days 1–5 after infection). In comparison, the rate of detection of NS1 antigen was 22% for secondary infection and 23% for primary infection (Fisher's exact test for NS1, p = 0.97 and, mean secondary IgG index = 3.8, mean primary IgG index = 2.1) at ≥11 days after onset of disease. Anti-DENV IgG antibodies levels at 1–5 days after onset of disease did not appear to inhibit NS1 Ag detection. The results, however, suggest that rise in levels of antibodies may be in part responsible for the lower NS1 detection rates (IgM-NS1 Pearson correlation, r = −0.62, IgG-NS1 Pearson correlation, r = −0.45). Thus, the association between rising levels of IgG, including factors such as IgG in antigenemia clearance and immune complex formation, with lower assay sensitivity needs to be further clarified. The differences between the detection rates in primary and secondary infection for each serotype were not statistically significant (primary and secondary DENV-1, chi-squared, p = 0.07, p = 0.24, p = 0.71, and p = 0.66 for DENV-2, DENV-3, and DENV-4, respectively).

Table 4. NS1 antigen positive rates by NS1 antigen ELISA in primary and secondary DENV infections
DayaPrimary infectionSecondary infection
 NS1 Ag (%)bNS1 Ag (%)
  1. Primary infection was defined by the positive detection of viral RNA when anti-DENV IgG antibodies were absent and anti-DENV IgM antibodies were absent or present. Secondary infection was defined by the presence of anti-DENV IgG antibodies when anti-DENV IgM antibodies were absent. Differences between positive detection rates of primary and secondary infection were not statistically significant (chi-squared test, p = 0.50).

  2. a

    Days after onset of disease.

  3. b

    Number of samples with indicated results/total number of samples (percentage of samples with indicated results, %). A total of 232 serum samples from patients with primary infection and 58 serum samples from patients with secondary infection was included ( Ntotal = 290 samples).

  4. c

    ND indicates that the value could not be determined due to an insufficient number of samples.

133/36 (92)5/5 (100)
222/27 (81)2/2 (75)
337/41 (90)10/11 (91)
421/24 (88)7/8 (88)
533/33 (100)6/6 (100)
1 to 5146/161 (91)30/32 (94)
613/14 (93)7/7 (100)
711/12 (92)2/4 (50)
89/11 (82)2/2 (100)
97/8 (88)0/2 (0)
103/4 (75)2/2 (100)
6 to 1043/49 (88)13/17 (76)
112/3 (67)1/3 (33)
120/1 (50)1/2 (50)
131/2 (50)0/0 (NDc)
≥142/16 (13)0/4 (0)
≥115/22 (23)2/9 (22)
Sum194/232 (84)45/58 (79)

Usage of Limited Amounts of Serum Sample in NS1 Antigen ELISA Assay

The utility of the NS1 antigen ELISA was assessed using limited amounts of serum samples: 5 and 0.5 μL (Table 5). Of 53 confirmed positive samples, 50 (94%) serum samples were positive by NS1 antigen ELISA using 5 μL of sample. When serum samples with a Biorad NS1 index value ≥10 were analyzed, NS1 antigen detection rates were 100% (37/37) using 5 μL of samples and 94% (31/33) with 0.5 μL (Table 5). When serum samples with a NS1 index value <10 were analyzed, detection rates were 81% (13/16) with 5 μL and 0% (0/10) with 0.5 μL. The differences between the NS1 antigen detection rates using 5 μL (1:10 dilution) of sample and undiluted samples were not statistically significant (Fisher's exact test, p = 0.24). In contrast, the differences were significant when 0.5 μL (1:100 dilution) was used (Fisher's exact test, p < 0.01, Table 5).

Table 5. NS1 antigen detection rate by NS1 Ag ELISA using diluted serum samples
Serum sample volume (dilution ratio to original test volume)No. of serum samples with indicated results/total number of samples (% positive)
  1. Samples consist of 53 confirmed DENV positive samples. The samples were positive for DENV NS1 antigen using 50 μL of sera.

  2. a

    Rate of detection was compared with those using undiluted serum samples by Fisher's exact test. Significant values were defined as p < 0.05.

  3. b

    Samples with an index value of ≥10 ( N = 37) as determined by NS1 antigen ELISA using 50 μL of serum samples.

  4. c

    Samples with an index value of <10 ( N = 16) as determined by NS1 antigen ELISA using 50 μL of serum samples.

Overall 
5 μL (1:10 dilution, p = 0.24)a50/53 (94%)
0.5 μL (1:100 dilution, p < 0.01)31/43 (72%)
(A) Index value ≥ 10b 
5 μL (1:10 dilution, p = 1.0)37/37 (100%)
0.5 μL (1:100 dilution, p = 0.49)31/33 (94%)
(B) Index value < 10c 
5 μL (1:10 dilution, p = 0.22)13/16 (81%)
0.5 μL (1:100 dilution, p < 0.01)0/10 (0%)

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

Widespread DENV transmission associated with international travel and urbanization continues to pose a global threat. As such, in addition to the current DENV diagnostic tools available, there is a tremendous need for reliable and dependable diagnostic tools that are relatively easy to use and that do not require highly skilled personnel or costly equipment. The dengue NS1 antigen ELISA is reported to be a promising tool for early dengue diagnosis.[13] While other investigators have reported the utility of various commercially available NS1 kits as a diagnostic tool for DENV infection,[14, 20-30] it is essential that their performance and utility be evaluated before their use becomes prevalent in different health sectors.[12] To determine the utility of the DENV NS1 assay for laboratory diagnosis of DENV infection of international travelers, we used serum samples from those who returned to Japan from various dengue endemic regions including Asia, Central and South America, Pacific Islands, and Africa. In combination with IgM ELISA, the detection of NS1 antigen in a single serum sample strongly suggests recent DENV infection.

The detection rate of NS1 was highest using samples from DENV1 patients as compared to detection rates (97%) of pooled serotypes (85%, Fisher's exact test, p < 0.01, days 1–10). However, the differences among the detection rates of DENV-2, DENV-3, and DENV-4 for days 1–5 and days 6–10 were not statistically significant. The presence of anti-DENV IgG antibody in the early phase of secondary infection did not appear to inhibit the detection of NS1 antigen (Table 4). NS1 antigen positive rates were at similar levels in primary and secondary infection. Thus, the ELISA method is useful in detection of viral antigens both in primary and secondary DENV infections. NS1 antigen positive rates were at high levels on days 1–5 and days 6–10. While some investigators found higher detection rates in primary infection as compared to secondary infection,[31-33] others found no difference in NS1 detection rates between primary and secondary infection[13, 34, 35] or higher detection rates in secondary as compared to primary infection.[36] Magnitude and kinetics of NS1 also varied with infecting serotype and viremia clearance.[37] Immune response in secondary patients also induces rapid rise of antibody titers and rapid clearance of DENV infection.[31, 37] However, the samples were evaluated in dengue hyper-endemic areas.[31, 32, 37] Strong humoral immune response may be induced during infection in dengue patients in endemic areas as compared to travelers from non-dengue endemic areas due to exposure to multiple infections which, in turn, result in a rapid rise of anti-NS1 antibodies and rapid antigenemia clearance. In our serum panel, the history of Japanese encephalitis and yellow fever vaccination of each traveler was not ascertained. Although anti-DENV IgG ELISA detects DENV-reactive IgG antibodies, other flavivirus IgG may cross-react with DENV. During secondary DENV infection (prior DENV exposure or sometimes after non-DENV flavivirus vaccination), antibody titers rise rapidly.[1] Our classification of primary and secondary patients is supported by the definition that IgG levels rise rapidly during secondary infection. In comparison, during primary infection, IgG levels are slow to rise. One of the DENV IgG ELISA assay limitations is the inability of the assay to distinguish between IgG of prior DENV exposure and non-DENV flavivirus vaccination. Thus, IgG antibodies secondary infection travelers may be induced by either DENV infection or past non-DENV vaccination. The ability of DENV cross-reactive antibodies that were induced by non-DENV vaccination or infection to influence NS1 antigenemia clearance and NS1 detection rate may be limited. Our results showed that NS1 levels decreased in both primary and secondary infection at the later phase of the disease (Table 4) with increasing levels of antibodies. There were no significant differences in NS1 antigen detection rates between primary and secondary infection at all phases of the disease (days 1–5, days 6–10, and ≥11 days) using our serum panel obtained from travelers. Further studies are required to address the role of antibodies induced by DENV infection and other non-DENV flavivirus vaccination (Japanese encephalitis virus, yellow fever virus) in NS1 detection and antigenemia clearance.

NS1 antigen has been detected concurrently with viremia and coincident with presence of disease symptoms.[38] We found that in travelers, while RT-PCR remains a highly sensitive method for the detection of viremia, positive rates by RT-PCR in the detection of DENV genome decreased after days 6–10 (detection rate range from 0–31%, Table 1). The results indicate that the positive detection rate using the NS1 ELISA is higher than that of RT-PCR for samples collected on and after days 6–10 and days ≥11. Confirmation of acute or early-phase DENV infection is of particular importance to imported dengue cases as disease surveillance data would be of significance to public health policies and regulations. Detection of NS1 by ELISA is thus useful in the early stages of the disease, particularly during the period of days 3–5 after onset of the disease, when viremia levels may be below detection levels and anti-IgM antibody levels have yet to rise.[14] Additionally, IgM ELISA is incapable of providing evidence of a recent infection as antibodies may persist for a few months after infection.[12] However, several characteristics of the NS1 antigen ELISA need to be addressed. These include waning assay sensitivity in the later phase of the disease (≥11 days, Figure 1). There were two samples that were RT-PCR positive but NS1 ELISA negative (Table 1). However, detection rate by RT-PCR was not significantly higher as compared to NS1 ELISA on days 1 and 2 (45/47 for RT-PCR, and 43/47 for NS1 ELISA, Fisher's exact test, p = 0.68, days 1–2 after infection). Thus, rather than as a replacement of conventional diagnostic methods, NS1 antigen ELISA could be used to increase the confidence of DENV infection diagnosis when performed in combination with IgM-ELISA and RT-PCR.[29, 39]

Using a subset of samples, we tested the NS1 antigen ELISA sensitivity with two different amounts of serum sample (5 and 0.5 μL). Using serum samples that tested positive for NS1 antigen by standard methods, detection rates were 94% with 5 μL and 72% with 0.5 μL (Table 5). However, the differences between the NS1 antigen detection rates using 5 μL (1:10 dilution) of sample and undiluted samples were not statistically significant (Fisher's exact test, p = 0.24). Thus, when using reduced serum volume, samples with NS1 positive results strongly suggest recent dengue infection and serum samples that were negative for NS1 require additional confirmatory diagnoses. However, the usage of reduced serum volumes would not be recommended when sufficient amount of samples are available. Although caution is required when using reduced volumes of serum samples, the method may prove to be invaluable when only limited amounts of sample are available.

One of the limitations of our study is that the samples (89%) were mostly obtained from Asian travelers from a nonendemic region to the Asian region. The study has, however, provided insights into the NS1 detection rates in travelers from a non-DENV endemic region, encompassing all four DENV serotypes and a broad range of immune profiles. NS1 rapid test has been proven useful in screening travelers in airports.[27, 40] Our study further extends utility of NS1 in dengue diagnosis in travelers[27, 40, 41] by using a broad range of patients with different immune profiles (primary and secondary) and serum samples obtained at different phases of disease.

The utility of the DENV NS1 antigen ELISA was assessed using serum samples obtained from returnees from dengue endemic regions including Asia, Central and South America, Pacific Islands, and Africa. In combination with other laboratory diagnostic tests such as anti-DENV antibody ELISA and RT-PCR, the detection of NS1 antigen in a single serum sample confirms recent dengue infection. The NS1 antigen ELISA demonstrated higher positive detection rates in the late phase of disease as compared to RT-PCR, and higher positive detection rates in the early phase of the disease as compared to IgM ELISA. These characteristics indicate that the assay may be useful even when either IgM ELISA or RT-PCR was negative. In combination with IgM-ELISA, the NS1 antigen ELISA increases the confidence of the diagnosis of recent DENV infection, particularly when only a single serum sample is available from a traveler who returned from dengue endemic areas.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

We would like to thank Mr. Kenichi Shibasaki for his expert technical assistance. We would also like to thank the health care practitioners of the clinics and hospitals in Japan for providing us with serum samples for laboratory diagnosis of dengue.

This work was supported by the funding from Research on Emerging and Re-emerging Infectious Diseases by the Ministry of Health, Labor and Welfare, Japan (grants H20-shinkou-ippan-015, H21-shinkou-ippan-005 and H23-shinkou-ippan-010).

Declaration of Interests

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References

The authors state they have no conflicts of interest to declare.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Declaration of Interests
  8. References
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