Vickers et al.

Journal of Biomedical Science (2015) 22:55

DOI 10.1186/s12929-015-0164-9

RESEARCH Open Access

The performance of the SD BIOLINE Dengue

DUO® rapid immunochromatographic test kit
for the detection of NS1 antigen, IgM and IgG
antibodies during a dengue type 1 epidemic in
Jamaica
Ivan E. Vickers1*, Kevin M. Harvey2, Michelle G. Brown1, Kereann Nelson1, Marion Bullock DuCasse2
and John F. Lindo1

Abstract
Background: Dengue is an important mosquito-borne viral infection that affects millions of persons worldwide.
Early diagnosis is necessary to effect appropriate management and decrease mortality. Immunochromatographic tests
are advantageous in producing dengue test results within 30 min but these results should be sensitive and specific. In
this study we evaluated the diagnostic performance of the SD BIOLINE Dengue DUO® rapid immunochromatographic
test kit. A panel of 309 dengue and 30 non-dengue single serum samples characterized by using reference
enzyme-linked immunosorbent assays (ELISAs) was used. These samples were received in the virology laboratory
for routine testing during a dengue type 1 outbreak between October to December, 2012.
Results: The overall diagnostic sensitivities of the SD BIOLINE Dengue DUO® rapid testfor IgM, IgG and NSI were
49.3 % (95 % CI: 41.3-57.4), 39.1 % (95 % CI: 33.3-45.2) and 90 % (95 % CI: 82.1-94.7), respectively. The IgM and
IgG detection rates were significantly lower than that of the NSI (p < 0.001). However the combination of the
IgM detection with NS1 detection or both NS1 and IgG resulted in a significant (p < 0.001) increase in sensitivity
to 97.5 % (95 % CI: 92.9-99.2) and 98.9 % (95 % CI: 96.0-99.7), respectively. These higher sensitivities were achieved
without any decrease in specificities.
Conclusions: This study revealed that combining two or more parameters of the SD BIOLINE Dengue DUO®
rapid kit significantly improved the sensitivity of diagnosis of dengue virus infection and supports its usefulness
in the Jamaican setting.
Keywords: Dengue, IgM, IgG, NS1, Antibody, Antigen, Sensitivity, Specificity, Positive predictive value, Negative
predictive value

* Correspondence: [email protected]
1
Department of Microbiology, The University of the West Indies, Kingston 7,
Mona, Jamaica
Full list of author information is available at the end of the article

© 2015 Vickers et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Vickers et al. Journal of Biomedical Science (2015) 22:55 Page 2 of 7

Background Hospital of the West Indies (UHWI), a tertiary referral

Dengue is a viral infection caused by the dengue virus hospital, after ethical approval was obtained (ECP 181,
(DENV). DENV is a member of the Flaviviridae family 12/13). The virology laboratory is the reference labora-
of ribonucleic acid (RNA) viruses which also includes tory for testing dengue virus in Jamaica and receives
West Nile virus (WNV), Yellow fever virus (YFV) and specimens from all 14 parishes of the island.
Japanese encephalitis (JE) virus [1, 2]. There are four se-
rotypes of dengue virus (DENV-1, DENV-2, DENV-3 Study design
and DENV-4) although recently a possible fifth serotype A retrospective cross sectional design was used to screen
(DENV-5) was reported [3]. The virus is arthropod archived single serum samples received in the virology la-
borne and is transmitted to humans by the bite of an in- boratory with a request for dengue IgM antibody testing
fected female mosquito. The primary vector is the Aedes between October and December 2012. All samples were
aegypti mosquito but other species such as Aedes albo- stored at −70 °C after routine diagnostic testing until in-
pictus and less commonly Aedes polynesiensis can also cluded in this study for evaluation. The inclusion criteria
transmit the virus [4, 5]. Dengue occurs in tropical and for the sample selection were: presence of the date of onset
subtropical regions of the world with endemicity in over of symptoms, presence of the date of collection of specimen
100 countries including Jamaica [2, 6–8]. Although den- and sufficient sample volume. A total of 339 of the 3402 ar-
gue is endemic in the Americas, outbreaks generally chived single serum samples met the inclusion criteria and
recur with a 3 to 5 year cycle [8]. The last epidemic in were selected. Demographic and clinical information were
Jamaica was in the year of 2012 and was caused by extracted from the hospital records.
DENV-1 [9, 10].
The clinical manifestations of dengue usually follow an Dengue diagnostic tests
incubation period of 2–7 days and may include a wide The dengue NS1 antigen ELISA (Standard Diagnostics Inc.,
variety of signs and symptoms [11]. According to the Seoul, Korea) and the dengue IgM and IgG antibody cap-
most recent classification by the World Health ture ELISAs (Focus Diagnostics, Cypress, PA, USA) were
Organization (WHO) persons are classified as having used as the reference methods [16–18]. All reference test-
dengue with or without warning signs or severe dengue ing procedures were performed and interpreted according
[12]. The criteria for dengue without warning signs in- to the manufacturers’ instructions except for the interpret-
clude fever and two of nausea and vomiting, rash, aches ation of the IgM assay. The IgM ELISA was interpreted
and pains, leucopenia and a positive tourniquet test. as: − positive: index value ≥1.2; negative: index value <1.0;
Warning signs include abdominal pain or tenderness, equivocal: index value >1.0 and <1.2. Samples (n = 28) that
persistent vomiting, mucosal bleeding, among others. were repeatedly equivocal were excluded from analysis. The
There is no vaccine or specific treatment for dengue but manufacturer’s instructions for the SD BIOLINE Dengue
early diagnosis and supportive management can decrease DUO® (SDB DD) NS1 Ag and IgG/IgM ICT were followed
the mortality of severe dengue disease [13]. and are described previously [19]. Briefly, 100 μl and 10 μl
The laboratory diagnosis of dengue includes virus iso- of serum specimen were added to the sample well “S” of
lation, serological and molecular techniques [5, 12, 14]. the NS1 Ag and IgM/IgG strips of the combo device, re-
Viral isolation is generally time-consuming while mo- spectively. Four drops of assay diluents were added to the
lecular methods are expensive. Enzyme-linked immuno- assay diluent well of the latter. Both strips of the device
sorbent assay (ELISA) is most often used in the were read at 15–20 min.
diagnosis of dengue in Jamaica and other countries.
These tests detect dengue specific antibodies such as im- Classification of samples
munoglobulin (Ig)-M, IgG, IgA or dengue antigens par- The samples were classified as dengue, non-dengue, pri-
ticularly non-structural (NS)-1 glycoproteins [15, 16]. mary, secondary, acute or convalescent. A sample was
More recently, rapid immunochromatographic tests defined as dengue if it was positive for at least one of
(ICTs) have become available. The diagnostic perfor- dengue IgM, IgG or NS1 antigen biomarker and non-
mances of the dengue ICT kits have been noted to vary dengue if negative for all three biomarkers. A primary
with different countries. We, therefore, sought to deter- dengue sample was one in which the IgM/IgG optical
mine the performance characteristics of a rapid dengue density (OD) ratio is ≥1.2. If the ratio was <1.2 it was
ICT kit in Jamaica. designated as secondary [12, 20, 21]. Sample collection
time was expressed in days(s) post onset of symptoms
Methods (DPO) and was interpreted with day 1 being the first
Study site 24 h. An acute sample was one with a DPO ≤ 5 days
The study was conducted at the virology laboratory in while a convalescent sample was defined as having a
the Department of Microbiology of the University DPO > 5 days. The combination strategies for the SDB
Vickers et al. Journal of Biomedical Science (2015) 22:55 Page 3 of 7

DD ICT are as described before [22]. An IgM/NS1 com- NS1 were 49.3 % (95 % CI: 41.3-57.4), 39.1 % (95 % CI
bination result meant dengue if either IgM or NS1 was 33.3-45.2) and 90 % (95 % CI: 82.1-94.7), respectively.
positive and non-dengue if both were negative irrespect- The IgM and IgG detection rates were significantly
ive of the IgG result. The IgM/NS1/IgG combination lower than that of the NS1 (p < 0.001). However, the
result was interpreted as dengue if at least one of SD combination of the IgM detection with NS1 detection
IgM, IgG or NS1 was positive and non-dengue if all or both NS1 and IgG resulted in a significant
three were negative. (p < 0.001) increase in sensitivity to 97.5 % (95 % CI:
92.9-99.2) and 98.9 % (95 % CI: 96.0-99.7), respectively.
Statistical analysis These higher sensitivities were achieved with increase
Microsoft Excel (Microsoft Inc., Redmond, Washington, specificities of 100 %. The kappa coefficients for the
USA) was used for data entry and data analysis was done NS1, IgM/NS1 and IgM/NS1/IgG parameters were 0.91
using Epi info 7 (Centers for Disease Control and Prevention, (95 % CI: 0.81-1.0), 0.97 (95 % CI: 0.86-1.0), and 0.96
Atlanta, USA). Chi-square test and the Fisher’s exact test (95 % CI: 0.83-1.0), respectively.
(two sided) were used to compare categorical variables. A The IgG test had the lowest sensitivity of 39.1 %
probability value (p) of < 0.05 was considered statistically (95 % CI: 33.3-45.2) but the highest individual specifi-
significant. The sensitivity, specificity, positive predictive city of 100 % (95 % CI: 95.6-100). It also demon-
value (PPV), negative predictive value (NPV) and kappa strated a very high individual positive predictive value
coefficient were calculated as previously described (PPV) of 100 % (95 % CI: 95.6-100) but a very low
[23, 24]. negative predictive value (NPV) of 34.7 % (95 % CI:
29.0-41.0). The PPV and NPV for the NS1 compo-
nent, the combined IgM/NS1 and IgM/NS1/IgG were
Results all high.
Patients’ samples
The 339 consecutively archived samples selected for the
evaluation were obtained from 159 (47 %) female and Sensitivity of SDB DD tests according to primary and
180 (53 %) males. The median age was 13 years (range, secondary dengue immune status
10 days to 95 years) and the median time between the Figure 1 shows that the IgM detection was signifi-
onset of illness and collection of specimens was 4 days cantly higher in samples with primary dengue (72.6 %;
(range, 1 to 22 days). The samples were characterized 95 % CI: 60.4-82.1 %) than secondary dengue (31.7 %;
using the reference ELISAs for dengue IgM, IgG and 95 % CI: 22.7-42.4) infection (p < 0.001). Although the
NS1 antigen and classified initially as 309 (91 %) dengue sensitivities of the NS1 and IgG markers were lower
and 30 (9 %) non-dengue. The 309 dengue samples were in secondary dengue samples (88.2 % and 52.1 %,
further defined by dengue phase as 189 (61 %) acute, respectively), than in primary (91.1 % and 63.0 %,
120 (39 %) convalescent and by immune status as 80 respectively), the differences were not significant.
(26 %) primary and 229 (74 %) secondary. There was a slight non-significant increase in the sen-
sitivities of the IgM/NS1 and IgM/NS1/IgG combined
Overall diagnostic performance dengue biomarkers in secondary dengue samples
The diagnostic performance of the individual assay (98 %; 95 % CI: 89.5-99.7 and 98.6 %; 95 % CI: 92.9-
parameters in comparison to the reference standard 99.8, respectively), over primary dengue samples
ELISAs is shown in Table 1. The overall diagnostic (97.1 %; 95 % CI: 90.2-99.2 and 98.6 %; 95 % CI: 92.3-
sensitivities of the SDB DD rapid test for IgM, IgG, and 99.0, respectively).

Table 1 Overall diagnostic performance of the SDB DD ICT against reference ELISAs
Dengue Parameter No. of dengue No. of SDB DD Sensitivity % Specificity % PPV % NPV %
ELISA ICT (95 % CI) (95 % CI) (95 % CI) (95 % CI)
pos/neg pos/neg
IgM 145/194 71/187 49.3 (41.3-57.4) 95.9 (92.1-97.9) 89.9 (81.3-94.8) 71.9 (66.2-77.0)
NS1 90/249 81/247 90.0 (82.1-94.7) 99.2 (97.1-99.8) 97.6 (91.6-99.3) 96.5 (93.5-98.1)
IgG 256/83 100/83 39.1 (33.3-45.2) 100 (95.6-100) 100 (96.3-100) 34.7 (29.0-41.0)
IgM/NS1 121/157 117/157 97.5 (92.9-99.2) 100 (97.6-100) 100 (96.8-100) 98.1 (94.6-99.4)
IgM/ NS1/ IgG 181/29 179/29 98.9 (96.0-99.7) 100 (88.3-100) 100 (97.9-100) 93.6 (79.3-98.2)
95 % CI is shown in parenthesis. pos = positive; neg = negative; SDB DD = SD BIOLINE Dengue DUO®; ICT = Immunochromatographic test
Vickers et al. Journal of Biomedical Science (2015) 22:55 Page 4 of 7

Primary dengue samples (n=80) Secondary dengue samples (n=229)

100.0

90.0
*
80.0
Sensitivity % (95% CI)
70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0
IgM NS1 IgG IgM/NS1 IgM/NS1/IgG
Dengue biomarkers
Fig. 1 Sensitivity of SDB DD ICT according to dengue immune status. Data represent sensitivity (%) and error bars represent 95 % CI. *indicates
significance at p < 0.001, when comparing primary and secondary dengue samples

Sensitivity of SDB DD tests according to acute and samples. It was hypothesized that dengue viral antigens,
convalescent phases inclusive of NS1, may form immune complexes with
The IgM was significantly more sensitive (p < 0.001) in high levels of dengue IgG antibodies and thus become
convalescent serum samples (67.2 %; 95 % CI: 55.3-77.2) undetectable [29, 30]. High titres of IgG are more likely
as compared to acute samples (33.8 %; 95 % CI: 24.2- to be found in secondary dengue infections. Andries
44.9) (Fig. 2). In contrast, the sensitivity of the IgG, NS1, et al. [28], for example, found significant reduction of
and the combined strategies of IgM/NS1 and IgM/NS1/ NS1 sensitivity in secondary infection (43.4 %) when
IgG were not significantly affected by acute or convales- compared to primary infection (89.5 %) (p < 0.001).
cent dengue phases. While the sensitivity of the SDB DD NS1 in this study
was generally lower in the presence of measurable IgG
Sensitivity of SDB DD NS1 test according to IgM/IgG antibodies this was not statistically significant. Similarly,
antibody profiles although we found a lower NS1 sensitivity in secondary
In this study, the presence of IgM and/or IgG did not infections than in primary infections, this again was not
significantly impact the sensitivities of the SDB DD NS1 significant. Our results, however, are not unique and
tests although the sensitivities generally trend higher in similar findings were reported by Gan et al. and
the absence of IgM/IgG (Table 2). The sensitivities of Sanchez-Vargas et al. [25, 26].
the SDB DD dengue biomarkers were not affected by It appears therefore that although IgG antibody titres
age or sex. may have some impact on the NS1 sensitivity levels, this
alone may not explain the low sensitivity levels obtained
Discussion in some studies [22, 27, 28]. Perhaps, other factors including
The NS1 test component of the SDB DD ICT kit had the type of reference method used in the comparisons
the highest individual sensitivity of 90 % when compared were contributing to the observed trends. Those studies
to the reference ELISA for the diagnosis of dengue in with low NS1 sensitivities all used non-NS1 comparators
Jamaica. Slightly lower but similarly high sensitivities such as virus isolation and genome detection by poly-
(81.6 % and 87.5 %) were also reported by Gan et al. in merase chain reaction (PCR) as the reference standards
Singapore [25] and by Sanchez-Vargas et al. in Mexico [22, 27, 28]. Hang et al. and Tricou et al. in their evaluations
[26], respectively. In both of those studies the authors of the relationship between NS1 sensitivity to viraemia
used a different NS1 ELISA (Platelia™) as comparator. using several NS1 assays have shown that NS1 levels
In contrast, others have reported markedly lower SDB correlate with viraemia [30, 31]. They have also found
DD NS1 sensitivity findings in the range of 44-51 % [22, that NS1 negative patients had significantly lower mean
27, 28]. The lower sensitivities were attributed to the viraemia than NS1 positive patients. Although the cor-
possible presence of high IgG antibody titres in those relation between NS1 detection and viraemia is not
Vickers et al. Journal of Biomedical Science (2015) 22:55 Page 5 of 7

Acute dengue samples (n=189) Convalescent dengue samples (n=120)

100.0

90.0

Sensitivity % (95%CI) 80.0

70.0

60.0

50.0 *
40.0

30.0

20.0

10.0

0.0
IgM NS1 IgG IgM/NS1 IgM/NS1/IgG
Dengue biomarkers
Fig. 2 Sensitivity of SDB DD ICT according to sample collection phase. Data represent sensitivity (%) and error bars represent 95 % CI. *indicates
significance at the p < 0.001 level when comparing acute and convalescent dengue samples

precise, we are of the opinion that studies which use previously published report of a sensitivity of 47 %
virus isolation or genome detection methods may be de- and 49.7 % in Puerto Rico and Malaysia, respectively
tecting lower levels of viraemia in those samples which, [17]. Clinicians should, therefore, not use a negative
in effect, are less likely to be NS1 positive. IgM result alone to rule out dengue infection in
The fact that the SD NS1 sensitivity, in our study, was these settings. The SDB DD dengue IgM test param-
high and was not significantly affected by factors such as eter was significantly lower in acute and secondary
primary/secondary infection status, acute/convalescent dengue samples. These are expected findings which
and the presence/absence of IgM/IgG antibodies is def- demonstrate the dynamics of the host IgM immune
initely advantageous. Accordingly, this test is ideal for response [29, 32]. IgM is therefore considered more
use in dengue endemic as well as non-endemic coun- a marker of recent infection rather than of acute in-
tries. It would also be extremely helpful in making den- fection [29] and so this limits its usefulness when
gue diagnosis in areas where patients present in either used alone as a single biomarker in the diagnosis of
the acute (≤5 days) and early convalescent (>5 days) dengue infection.
phases of dengue illness. The limitations of the SDB DD IgM biomarker alone,
The overall sensitivity of the SDB DD IgM compo- however, disappeared when it was combined with other
nent alone was low and is consistent with a dengue biomarkers. Herein lies the advantage of the
SDB DD kit in that it has the capacity to measure both
Table 2 Sensitivity of the SDB DD NS1 parameter according to dengue antigen and dengue antibodies in one test. This
IgM/IgG antibody profile allows for different testing strategies. We explored differ-
Dengue antibody Sensitivity % N P value ent combination approaches with the IgM/IgG and NS1
profile (95 % CI) biomarkers and found that combining any two or more
IgM−/IgG− 94.4(74.2- 99.0) 18 0.899a of the individual components resulted in even greater
+
IgM /IgG −
91.2(77.0-97.0) 34 levels of diagnostic sensitivities. Our data provides evi-
−
IgM /IgG +
100(75.8-100) 12 0.265b dence, in keeping with others who have recently re-
+ + ported, that using a combination strategy enhances the
IgM /IgG 80.8(62.1-91.5) 26
overall diagnostic performance of rapid dengue diagnos-
95 % CI is shown in parenthesis; − = negative; + = positive; a = comparison
between IgM−/IgG− and IgM+/IgG− ; b = comparison between IgM−/IgG+
tic kits [19, 25–28, 33]. For example, Wang et al. [19] re-
and IgM+/IgG+ ported improvement in sensitivity from 53.5 % (IgM
Vickers et al. Journal of Biomedical Science (2015) 22:55 Page 6 of 7

alone) to 88.7 % (NS1/IgM) while Sanchez-Vargas et al. Acknowledgements

[26] found enhanced sensitivity from 60.51 % (IgM This study was partially funded by the Ministry Of Health (MOH), Jamaica
and USAID PEPFAR Project. We wish to also thank the members of staff of
alone) to 90.65 % (NS1/IgM/IgG). It should be noted the MOH and the virology laboratory (UHWI) for their kind support.
that in both of the above mentioned studies the im-
provement in sensitivities came with slight decrease in Author details
1
Department of Microbiology, The University of the West Indies, Kingston 7,
specificities unlike our study in which there were slight Mona, Jamaica. 2Ministry of Health, Kingston, Jamaica.
increase in specificities to 100 %. This pattern of im-
provement in test performance by combination of mul- Received: 16 February 2015 Accepted: 3 July 2015

tiple dengue biomarkers is not only observed for SDB

DD ICT kits but also for other rapid dengue diagnostic
kits [25, 33]. Additionally, in the current study, the References
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