Electronic Supplementary Materials

(Online Resource 1 to 7)

Association between dengue severity and plasma levels of dengue-specific IgE and chymase

Miho Inokuchi1†, Shyam Prakash Dumre1†, Shusaku Mizukami1, Mya Myat Ngwe Tun2, Mohamed Gomaa
Kamel3, Dao Huy Manh1, Le Hong Phuc4, Nguyen Van Thuong5, Tran Van An4, Lan Nguyen Weiss5,
Tarek Turk6, Tran Ngoc Dang7, Vu Thi Que Huong5, Kouichi Morita2, Nguyen Tien Huy8,*, Kenji
Hirayama1,*

Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

1

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

2

Faculty of Medicine, Minia University, Minia 61519, Egypt

3

Nguyen Dinh Chieu Hospital, Ben Tre Province, Vietnam

4

Department of Immunology and Microbiology, Pasteur Institute, Ho Chi Minh City, Vietnam
5

Faculty of Medicine, Damascus University, Damascus, Syria

6

Department of Environmental Health, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam
7

Department of Clinical Product Development, Institute of Tropical Medicine, Nagasaki University,

8

Nagasaki, Japan
Authors equally contributed the work.
†

Correspondence to Kenji Hirayama (E-mail: hiraken@nagasaki-u.ac.jp)

or Nguyen Tien Huy (E-mail: tienhuy@nagasaki-u.ac.jp)
Department of Immunogenetics or Department of Clinical Product Development, Institute of Tropical
Medicine, Nagasaki University, Nagasaki 852-8523, Japan
Tel: +81-95-819-7820

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 2.6

2.4419
2.4

2.2

2.0522 2.0355
2 1.9732

1.8074 1.8376
1.8 1.7891
1.7000
1.6 1.6069

1.4

1.2

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110µg/ml 82.5µg/ml 55µg/ml

20F(750x) 20F(1000x) 20F(1500x)

Figure 4.5.b 20F P/N ratio

Online Resource 1. Optimization of in-house capture ELISA system for DENV-specific IgE. In the
figure, Y-axis denotes the P/N ratio of DENV specific IgE and X-axis denotes the different concentrations
of capture antibody. Different dilutions of detection antibody are indicated by different colored lines in
the linearity plotting. P/N ratios is the ratio of optical density (OD) of sample and OD of negative control.
20F is sample ID. DENV, dengue virus.

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Online Resource 2. Kinetic profiles of IgEs and plasma chymase in dengue and OFI patients,
Vietnam. The dynamics of each parameter for individual patient is shown by thin lines (different colors
for Level 1, 2, 3, and OFI patients), and the median of each group by smooth bold lines (trend lines).
Values at post-discharge (Post-dis) are plotted with dots and their medians are shown by short bold lines.
*indicates significant difference by bootstrapping test for A: DENV specific IgE (between Level 2 and
OFI on day -1; between each level and OFI on both day 0 and post-discharge), B: total IgE (at post-
discharge between Level 3 and OFI), C: S/T ratio (between Level 1 and 3, on day -2; between Level 3
and OFI on day 0; between Level 1 and 3, and between each level and OFI at post-discharge) and D:
chymase (on day 0 between Level 3 and OFI and at post-discharge between Level 1 and 2). OFI, other
febrile illness; DENV, dengue virus; S/T ratio, DENV specific IgE/total IgE ratio

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Online Resource 3. DENV specific IgE in patients with different levels of dengue illness and OFI patients, Vietnam

Number of patients (n) Comparison between different levels and OFI (95% CI)
Time points
Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
Day -3 6 4 2 1 (-0.35−0.08) <

Day -2 12 9 7 2 (-0.17−0.17)< (-0.45−0.142)< (-0.51−0.24)<

Day -1 15 9 6 5 (-0.69−0.45)< (-1.67−0.56)< (-0.52−1.05)< (-0.03−0.59)> (0.04−1.04)>* (-0.06−0.79)>
Day 0 16 9 6 7 (-0.66−0.44)< (-0.66−0.67)< (-0.48−0.61)> (0.20−1.17)>* (0.43−1.43)>* (0.40−1.39)>*
Day 1 12 3 1 4 (-0.23−1.02)>
Day 2 6 2 1 2 (-1.03−0.96)<
Day 3 3 4 2 2 (-0.96−0.60)<
Post-dis 16 9 8 5 (-0.48−0.69)< (-0.80−0.32)< (-0.86−0.62)< (0.89−1.17)>* (0.38−1.52)>* (0.75−1.82)>*
DENV, dengue virus; OFI, other febrile illness; CI, confidence interval; Post-dis, post-discharge
Difference in plasma DENV specific IgE among different levels of dengue illness and OFI was analyzed by bootstrapping test with 1000 replications. When the sample
size was < 3 in any group, statistical difference was not assessed.
>
indicates first group > second group; <indicates second group > first group between the comparison groups.
*indicates significant difference.
The figures in the parentheses indicate 95% CI unless otherwise indicated.

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Online Resource 4. Plasma total IgE in patients with different levels of dengue illness and OFI, Vietnam

Number of patients (n) Comparison between different levels and OFI (95% CI)
Time points
Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
Day -3 6 4 2 1 (-11702−2346)<
Day -2 12 9 7 2 (-2559−1817)> (-846−2041)> (-1392−3215)>
Day -1 15 9 6 5 (-7236−1945)< (-1812−2424)> (-1767−6342)> (-3224−1370)> (-3236−4150)> (-3657−1527)<
Day 0 16 9 6 7 (-761−2460)> (-1717−3179)> (-1805−6012)> (-1149−2404)> (-1528−6039)> (-2047−1687)<
Day 1 12 3 1 4 (-7375−2225)< (-1407−1668)< (-1765−7071)>
Day 2 6 2 1 2
Day 3 3 4 2 2 (-6261−2250)< (-3042−3125)<
Post-dis 16 9 8 5 (-2510−967)< (-206−1408)> (-341−2968)> (-3092−484)> (-3363−588)> (-3497−-266)<*
OFI, other febrile illness; CI, confidence interval; Post-dis, post-discharge
Difference in plasma total IgE among different levels of dengue illness and OFI was analyzed by Bootstrapping test with 1000 replications. When the sample size was
< 3 in any group, statistical difference was not assessed.
>
indicates first group > second group; <indicates second group > first group between the comparison groups.
*indicates significant difference.
The figures in the parentheses indicate 95% CI unless otherwise indicated

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Online Resource 5. DENV specific IgE/total IgE ratio (S/T ratio) in patients with different levels of dengue illness and OFI, Vietnam

Time Number of patients (n) Comparison between different levels and OFI (95% CI)
points Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
Day -3 6 4 2 1 (-1.72−4.22) >

Day -2 12 9 7 2 (-3.20−0.51)< (-2.20−-0.51)<* (-2.37−1.84)<

Day -1 17 9 6 5 (-3.75−1.58)< (-3.50−1.02)< (-3.36−3.20)< (-1.21−3.15)> (-1.15−4.81)> (-0.70−3.86)>
Day 0 16 9 6 7 (-6.57−2.78)< (-4.74−0.33)< (-5.22−4.17)< (-0.47−3.84)> (-0.45−8.75) (0.76−6.92)>*
Day 1 13 3 1 4 (-3.13−5.95)> (-2.39−5.14)> (-4.51−6.45)>
Day 2 6 2 1 2 (-11.22−4.04)<
Day 3 3 4 2 2 (-8.60−8.07)>
Post-dis 16 9 8 5 (-6.19−5.27)< (-6.97−-0.02)<* (-6.39−3.16) (1.94−8.90)>* (3.36−9.61)>* (6.44−11.95)>*
DENV, dengue virus; OFI, other febrile illness; CI, confidence interval; Post-dis, post-discharge
Difference in plasma S/T ratio among different levels of dengue illness and OFI was analyzed by Bootstrapping test with 1000 replications. When the sample size was
< 3 in any group, statistical difference was not assessed.
>
indicates first group > second group; <indicates second group > first group between the comparison groups.
*indicates significant difference.
The figures in the parentheses indicate 95% CI unless otherwise indicated.

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Online Resource 6. Plasma chymase concentration in patients with different levels of dengue illness and OFI, Vietnam

Time Number of patients (n) Comparison between different levels and OFI (95% CI)
points Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
Day -3 6 4 3 1 (-7.2−3.33) <
(-3.89−3.59) <
(-4.71−5.99) >

Day -2 13 9 7 2 (-5.25−1.83) <

(-2.15−3.57) >
(-0.91−5.26)>
Day -1 16 9 5 5 (-1.85−1.62)> (-1.60−2.71)> (-2.28−3.72)> (-14.02−0.86)< (-14.77−0.08)< (-14.85−-0.001)<
Day 0 17 9 6 7 (-5.25−0.51)< (-3.26−1.96)> (-0.76−6.76)> (-9.20−0.36)< (-6.85−4.32)< (-10.11−-0.09)<*
Day 1 12 3 1 4 (-2.97−4.17)> (-17.77−3.37)< (-19.71−5.26)< (-19.85−1.31)<
Day 2 6 2 1 2
Day 3 3 4 2 2 (-12.11−7.1)<
Post-dis 16 9 8 5 (-25.78−-0.14)<* (-10.10−4.75)< (-4.45−21.00)> (-8.45−3.67)< (-2.91−16.36)> (-7.07−9.14)>
OFI, other febrile illness; CI, confidence interval; Post-dis, post-discharge
Difference in plasma chymase concentration among different levels of dengue illness and OFI was analyzed by Bootstrapping test with 1000 replications. When the
sample size was < 3 in any group, statistical difference was not assessed.
>
indicates first group > second group; <indicates second group > first group between the comparison groups.
*indicates significant difference.
The figures in the parentheses indicate 95% CI unless otherwise indicated.

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 Online Resource 7. Plasma chymase concentration in patients with different levels of dengue illness and OFI during early acute phase (-48 h
to-6 hours), Vietnam
Number of patients (n) Comparison between different levels and OFI (95% CI)
Time point
Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
-48 to -6 hrs 15 10 7 4 (-4.43−1.23) <
(-1.84−2.41) >
(-0.93−4.16) >
(-6.32−0.86) <
(-5.05−2.52) <
(-6.34−0.57)<

Online Resource 8. DENV specific IgE, total IgE and their ratio (S/T ratio) in patients with different levels of dengue illness and OFI during early acute phase
(-48 h to -6 h), Vietnam
Comparison between different levels and OFI (95% CI)
Time Number of patients (n)
IgE type
point
Level 1 Level 2 Level 3 OFI Level 1 vs 2 Level 1 vs 3 Level 2 vs 3 Level 1 vs OFI Level 2 vs OFI Level 3 vs OFI
DENV
13 10 7 4 (-0.4−0.499)> (-0.74−0.55)< (-0.73−0.47)< (-0.08−0.56)> (-0.04−0.6)> (-0.06−0.76)>
-48 to specific IgE
-6 hrs Total IgE 13 10 7 4 (-4369−2833)> (-1094−3598)> (-1413−4825)> (-3182−2434)< (-3627−2910)< (-4103−1250)<
S/T ratio 13 10 7 4 (-3.15−1.35)< (-4.28−-0.26)<* (-4.06−1.81)< (-1.40−2.57)> (-1.57−4.31)> (0.29−5.22)>*
In Online Resource 6 and 7, abbreviations used are: S/T ratio, DENV specific IgE/total IgE; OFI, other febrile illness; CI, confidence interval; Post-dis, post-discharge.
Difference among different levels of dengue illness and OFI was analyzed by Bootstrapping test with 1000 replications. >indicates first group >second group; <indicates
second group > first group between the comparison groups. The figures in the parentheses indicate 95% CI unless otherwise indicated.

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Online Resource 9. Patient details of acute phase samples collected (-48 h to-6 hours) based on fever onset day

Number of patients sampled (based on onset of fever day)

Severity Day 2 Day 3 Day 4 Day 5 Total

Level 1 1 9 3 2 15

Level 2 2 3 0 1 10

Level 3 0 5 0 2 7

Total 3 (10%) 17 (59%) 3 (10%) 5 (20%) 32 (100%)

Patient’s single sample was collected -6 to -48 hrs i.e. 0 to -2 day of defervescence (which corresponds to days 2 to 5 of fever onset in our cohort). Almost 70% (20/29)
of the patients in this analysis were sampled during 2 to 3 days of fever onset, 10 % (3/29) on day 4, and 20% (5/29) on day 5 of fever onset.

Online Resource 10. Protocol of the in-house ELISA to detect DENV specific IgE

Development of in-house ELISA system for detection of DENV specific IgE in plasma:

An in-house ELISA system was developed for measurement of DENV specific IgE in the plasma samples. ELISA conditions were optimized for all parameters prior to

testing with the study samples, and, a pilot assessment of refined optimization was performed with small number of plasma samples from dengue patients, OFI patients

and healthy individuals.

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First, F96 CERT Maxisorp NUNC IMMUNO plates (ThermoScientific, Massachusetts, USA) were coated with 100 µl of 110 µg/ml Goat Anti-human IgE (GenTex,

CA, USA), sealed and incubated overnight at 4°C. For blocking, 100 µl of 4% BlockAce (DS pharma biomedical, Osaka, Japan) prepared in phosphate-buffered saline

(PBS) was added into each well and incubated for 1 hour at 37°C. The wells were emptied and washed 4 times with PBS-Tween 20 by keeping 3 minutes incubation

with wash buffer during each wash step. 100 µl of plasma samples and negative control (samples from healthy individuals pre-confirmed negative for anti-DENV IgM

and IgG antibodies by ELISA) pre-diluted 1:100 in PBS+Tween 20 with NaN3 solution were added and incubated for 1 hour at 37°C. After sample incubation, plate

was washed 4 times as described before. 100 µl of undiluted dengue tetravalent antigen (25 unit each serotype × 4 serotypes) (prepared by the Virology Department,

NEKKEN as described previously) (Ngwe Tun et al., 2013) was added to each well and incubated for 1 hour at 37°C. Plate was washed 4 times to remove excess of

antigen and incubated for 1 hour at 37°C with 100 µl HRP conjugated anti-Flavivirus mouse monoclonal antibody (prepared by The Virology Department, NEKKEN

as described previously) (Ngwe Tun et al., 2013) diluted 1:1500 in PBS+Tween 20. After 1 hour, unbound conjugated antibody was washed away by 4 washes. The

plate was developed with 100 µl of 10 X diluted ortho-phenilenediamine (OPD) (SIGMA, MO, USA) in H 2O2 as substrate for 1 hour at room temperature in dark.

Finally, the reaction was stopped with 100 µl of 1 N H2SO4 and the plate was read at 492 nm.

DENV-specific IgE was measured as the ratio of optical density (OD) of plasma sample and negative control (NC) (OD of sample divided by OD of NC = P/N unit).

Therefore, a ratio of dengue specific IgE and total IgE (S/T ratio) was further calculated by the following formula:

S/T ratio = P/N ratio of sample wells

Total IgE OD of sample /OD of NC

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Reference: Ngwe Tun, M.M., Thant, K.Z., Inoue, S., Kurosawa, Y., Lwin, Y.Y., Lin, S., Aye, K.T., Thet Khin, P., Myint, T., Htwe, K., Mapua, C.A., Natividad, F.F.,

Hirayama, K., Morita, K., 2013. Serological characterization of dengue virus infections observed among dengue hemorrhagic fever/dengue shock syndrome cases in

upper Myanmar. J. Med. Virol. 85, 1258–1266. doi:10.1002/jmv.23577.

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