Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of

acute dengue infection

1 1 1
Rosario Vivek, PhD , Syed Fazil Ahamed, MSc , Shalini Kotabagi, MSc , Anmol Chandele,
2 3 3 2 4
PhD , Ira Khanna, MBBS , Navin Khanna, PhD , Kaustuv Nayak, PhD , Mary Dias, MD ,
2,5 1,6,*
Murali-Krishna Kaja, PhD , Anita Shet, MD PhD

PII: S0732-8893(16)30319-4
DOI: doi: 10.1016/j.diagmicrobio.2016.09.020
Reference: DMB 14203

To appear in: Diagnostic Microbiology and Infectious Disease

Received date: 18 May 2016

Revised date: 1 September 2016
Accepted date: 27 September 2016

International Vaccine Access Center, Johns Hopkins Bloomberg School of Public Health,
Baltimore, USA

E-mail address: ashet1@jhu.edu

4
ABSTRACT

The catastrophic rise in dengue infections in India and globally has created a need for an
accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated
the diagnostic performance of NS1/IgM RDT (Dengue Day 1) using 211 samples from a
pediatric dengue cohort representing all four serotypes in southern India. The dengue-positive
panel consisted of 179 dengue RT-PCR positive samples from symptomatic children. The
dengue-negative panel consisted of 32 samples from dengue-negative febrile children and
asymptomatic individuals that were negative for dengue RT-PCR/NS1 ELISA/IgM/IgG.
NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed
high sensitivity throughout the acute phase of illness, in primary and secondary infections, in the
different severity groups, and detected all the four serotypes, including co-infections. This
NS1/IgM RDT can be a useful point-of-care assay for rapid and reliable diagnosis of acute
dengue and an excellent surveillance tool in our battle against dengue.

KEYWORDS
Dengue, Diagnostics, Evaluation, NS1 protein, Rapid Diagnostic Test.

5
INTRODUCTION

Dengue is the most common arthropod-borne viral infection transmitted by mosquito to humans
that poses a tremendous public health problem. Rapid urbanization, increased travel and climate
change have contributed to the increased dengue cases seen globally and in India (Gupta et al.,
2012). Dengue virus (DENV) causes an estimated 96 million apparent infections a year globally,
and India alone is estimated to contribute 34% of the global total(Bhatt et al., 2013). However,
true disease burden based on active surveillance in India is not known. Only a small proportion
of dengue-infected symptomatic patients develop severe disease, while the majority recover after
a self-limiting illness or go through inapparent or subclinical infection. An early and accurate
diagnosis of dengue can lead to early intervention and better clinical management. Moreover, a
simple low-cost point-of-care test for dengue surveillance would be useful in assessing the true
burden of dengue infection in India.

Understanding the structure of dengue virus and the host immune response has contributed towards
development of a range of diagnostic tests. There are four distinct serotypes; DENV1, DENV2,
DENV3 and DENV4. All four serotypes have been shown to circulate in India (Chaturvedi and
Nagar 2008; Myers et al., 1970). DENV consists of 3 structural proteins and 7 non-structural
proteins. The non-structural protein 1 (NS1) is a glycoprotein released very early from infected cells
into blood(Smith and Wright 1985). Laboratory diagnosis based on viral isolation is highly specific
but poor in sensitivity, is labor-intensive and expensive. Use of reverse transcriptase polymerase
chain reaction (RT-PCR) may provide an early and accurate diagnosis, but requires specialized
equipment not always available in endemic settings. Serological diagnosis using anti-dengue IgM
and IgG antibodies is a useful diagnostic and surveillance tool, however the limitations are its cross-
reactivity with other flaviviruses, the need for paired sera for definitive diagnosis, and poor
sensitivity during the acute phase of infection when serum antibodies are below the limit of
detection (Calisher et al., 1989). Serological assays based on anti-dengue IgM and IgG also have
low specificity due to antigenic cross-reactivity and can have decreased utility for early diagnosis of
dengue. Dengue NS1 therefore is a useful tool for early diagnosis of dengue and an excellent target
for dengue assay development. An NS1 antigen capture enzyme linked immunosorbent assay
(ELISA) was developed for the first time in 2000 (Young et al., 2000). Since then, a number of
assays have been developed that

6
detect both NS1 antigen and IgM that has increased the diagnostic window period and sensitivity
particularly in secondary dengue infection (Blacksell et al., 2008; Fry et al., 2011; Gan et al.,
2014). The use of RDTs for point-of-care diagnosis in resource-limited and outbreak settings
underscores the need for more reliable and accurate NS1 based RDT assays. This study
prospectively evaluates the diagnostic performance and operational characteristics of an
indigenously developed dengue RDT (Dengue Day 1 Test, J Mitra & Co, New Delhi, India)
using clinical samples from a pediatric dengue cohort from southern India. In addition, an NS1
ELISA (DENV Detect NS1 ELISA, InBios International, Inc. USA) was also evaluated using
the same platform.

METHODS

Study setting and population: The current study is part of an ongoing collaborative pediatric
dengue cohort study between St Johns Medical College Hospital, Bangalore and International
Center for Genetic Engineering and Biotechnology (ICGEB), Delhi. Institutional ethical
approval was obtained from both institutions prior to patient recruitment. Children with
suspected or probable dengue who were admitted to St. Johns Medical College Hospital,
Bangalore from October 2014 to October 2015 were recruited for the study after obtaining
written informed consent. Clinical diagnosis of dengue was made using the WHO guidelines
("World Health Organization and UNICEF, Handbook for clinical management of dengue,
2012"). Patients suspected with acute dengue-like illness and NS1 / IgM positive were clinically
classified into mild dengue without warning signs (group A), dengue with warning signs (group
B) and severe dengue (group C) based on the recent WHO classification("World Health
Organization and UNICEF, Handbook for clinical management of dengue, 2012"). Blood
samples were collected from each patient on the first day of hospitalization, serum separated for
immediate RDT testing and retrospective RT-PCR and anti-dengue IgM/IgG by capture ELISA.

Laboratory reference tests: The presence of dengue virus and corresponding serotypes was
confirmed by RT-PCR and sequencing. Briefly, serum sample from each patient with clinically
diagnosed dengue was extracted for total RNA using the QIAamp Viral RNA kit (Qiagen,
Germany). Extracted RNA was converted to complementary DNA (cDNA) by reverse

7
transcriptase (Fermentas) using random hexamers (Proteogen). The cDNA was amplified using a
sequential algorithm of three modified PCR methods targeting the capsid premembrane (C-prM)
and envelope (Env) regions of the dengue genome in order to maximize DENV detection and
serotyping (Ahamed et al., 2016; Lanciotti et al., 1992; Yenchitsomanus et al., 1996). All PCR
products were sequenced and nucleotide sequence similarity searches were performed using
NCBI BLAST server on GenBank for DENV serotype confirmation. Dengue patients samples
positive by RT-PCR constituted the dengue positive panel. Healthy volunteers and febrile non-
dengue patients negative by RT-PCR and negative for anti-dengue IgM and IgG antibodies by
capture ELISA (J. Mitra & Co) comprised the dengue negative panel. Primary and secondary
infection status of the dengue positive panel was determined using dengue IgM & IgG capture
ELISA (PanBio, Product codes 01PE10/01PE20).

NS1/IgM Rapid Diagnostic Test (RDT): The Dengue Day 1 Test (J. Mitra & Co) is a rapid solid
phase immunochromatographic test with separate cassettes for the qualitative detection of dengue
NS1 antigen and differential detection of IgM and IgG antibodies to DENV in human serum. The
test was performed according to the manufacturers instructions. Briefly, 70 l of serum was added
into the sample well of the NS1 cassette and results read at 20 min. To the IgM/IgG antibody
cassette, 10 l of serum was added into the sample well and two drops of dengue antibody assay
buffer was added into the buffer well and results read at 20 min. Each cassette has a control and test
line; the appearance of both control and test lines indicated a positive result, and appearance of the
control line alone indicated a negative result. The test was repeated if the control failed or the result
was indeterminate. In this study NS1-only RDT results and combination of NS1 and/or IgM RDT
(NS1/IgM RDT) results have been considered for evaluation. IgG RDT results have not been
considered, as the presence of IgG antibodies alone may not be used for determining acute dengue
infection. The analysis of a single specimen can be completed in about 30 minutes, and a group of
10 specimens within 40 minutes.

NS1 ELISA: Patients sera were tested for dengue NS1 antigen using a sandwich immunoassay
DENV Detect NS1 ELISA (InBios, Seattle, WA, USA) according to manufacturers instructions.
The threshold value of the assay for a positive result was first calculated based on the mean optical
density (OD) values obtained with the cut-off controls read at 450 nm. Immune

8
status ratio (ISR) of a sample was then calculated from the ratio of the sample OD divided by
the mean OD of the cut-off controls. ISR 1 was considered positive for the presence of NS1
antigen. All controls were tested in duplicate, and sera with OD values close to cut-off (1.1 >
ISR > 0.9) were repeated in duplicate to verify sample status.

Statistical analysis: The NS1 ELISA, NS1 RDT and NS1/IgM RDT results were compared
against the dengue positive panel and dengue negative panel to determine their sensitivity,
specificity, positive predictive value and negative predictive value. Sub-group analyses were
performed to calculate the sensitivity and specificity of the RDT and ELISA for age, clinical
severity, days post-onset of fever, and dengue serotypes. Positive and negative predictive
values were calculated based on the prevalence of acute dengue infection in children
presenting to the hospital during the study period. Confidence intervals for sensitivity and
specificity were set at 95%, and a two-tailed p-value <0.05 was considered to be statistically
significant. All analyses were performed using Stata v13 (Statacorp).

RESULTS

A total of 211 samples were included in this study. The dengue positive panel comprised of
179 samples from children with acute symptomatic dengue confirmed by RT-PCR (Figure
1). The age of the children ranged from 4 months to 15 years (mean age 6.84.5 years) and
males constituted 61%. Febrile patients presented with a mean days post-onset of fever
(DPO) of 4 days (1.4) and all the dengue positive samples were stratified based on the
duration of illness represented by DPO. There were 111 (67.6%) primary and 53 (32.3%),
secondary infections within the dengue positive panel (8 samples were equivocal with an
IgM/IgG ratio of 1.2 and 7 samples did not have available plasma for serological analyses).

The serotype distribution in the 179 RT-PCR positive samples were as follows; DENV1
(95; 53.1%), DENV2 (46; 25.7%), DENV3 (21; 11.7%), DENV4 (3; 1.7%) and co-
infections with two or more serotypes (14; 7.8%). Mild dengue was seen in 2.8%, dengue
with warning signs among 57.0%, and severe dengue in 40.2%. The dengue negative panel
consisted of 32 samples from febrile and asymptomatic individuals. These included 11
children with febrile non-dengue
infection, and 21 healthy volunteers; all these samples were negative for dengue RT-PCR and
anti-dengue IgM and IgG by capture ELISA (Figure 1).
9
Figure 1: Flow diagram showing the panel used for the evaluation of NS1 ELISA and RDT
against the reference standard.

Febrile dengue cases evaluated

Healthy non-febrile
for this study= 233 subjects = 25

10
 RT- RT-PCR Negative = 54
PC
R
& excluded
seq
uen
cin
g RT-PCR, IgM & IgG anti-dengue
Pos negative by ELISA = 11
itiv
e=
17
9

Non
dengue
panel
used
for this
evaluat
ion
study
= 32
(Deng
Dengu
ue
e negati
confir ve
med panel)
cases
used
for
this
evalua
tion
study
= 179
(Deng
ue
positi
ve
panel)

11
 NS1/IgM RDT: Overall, the NS1/IgM RDT results showed 89.4% sensitivity
and 93.8% specificity. The positive predictive value (PPV) and negative
predictive value (NPV) was 98.8% and 61.2%, respectively (Table 1). The
sensitivity of NS1/IgM RDT in primary infections was 95.4% and in
secondary infections was 77.3%.

Table 1: Performance characteristics of Dengue Day 1 Test RDT and DENV

Detect NS1
ELISA against the reference standard*

Number Number
Sensitivity Specificity Positive Negative
with with
(%) (%) predictive predictive
Tests positive negative
value value
test test
(95% CI) (95% CI) (95% CI) (95% CI)
(n=179) (n=32)
Dengue Day 1 89.4 93.8 98.8 61.2
160 30
Test (NS1/IgM) (83.9 - 93.5) (79.2 - 99.2) (95.6 - 99.9) (46.2 - 74.8)
Dengue Day 1 82.7 96.9 99.3 50
148 31
Test (NS1 only) (76.3 - 87.9) (83.8 - 99.9) (96.3 - 100) (37.0 - 63.0)
DENV Detect 84.4 100 100 53.3
151 32
NS1 ELISA (78.2 - 89.3) (89.1 - 100) (97.6 - 100) (40 - 66.3)

* Dengue positive panel: Clinical dengue diagnosis based on 2012

WHO Classification, dengue RT-PCR positive; Dengue negative panel:
Non-dengue febrile illness or healthy volunteers; dengue RT-PCR
negative + NS1 ELISA, dengue IgM and IgG ELISA negative

12
The sensitivity of the NS1/IgM RDT was 100% on DPO 1 and DPO 2 and started to
drop slightly from DPO 3 (95.1%) through day DPO 5 (82.9%) and again the
sensitivity increased to 100% on DPO 6 and remained 100% through DPO 10 (Figure
2).

Figure 2: Sensitivity of Dengue Day 1 RDT and InBios NS1 ELISA stratified by days
post-onset of fever (DPO). The numbers in parenthesis below the x axis represents the
numbers of children considered at each time point.

Variations in serotype-based sensitivities were as follows: DENV1 (84.2%), DENV2

(95.7%), DENV3 (90.5%), DENV4 (100%) and co-infections with two or more serotypes
(100%) (Figure 3A). Sensitivity of NS1/IgM RDT for the three WHO classified clinical
severity groups A, B and C were 100%, 99% and 75%, respectively (Figure 3B). There
were no significant variations in sensitivity or specificity based on age or sex in this
cohort.

13
Figure 3A & 3B: Sensitivity of Dengue Day 1 NS1/IgM RDT, Dengue Day 1 NS1
RDT and InBios NS1 ELISA among different serotypes (3A) and severity groups
(3B).

When only the NS1 component of the RDT was considered, the overall sensitivity was
82.7% and the specificity was 96.9% (Table 1). In primary infections the sensitivity was
90% and 67.9% in secondary infections. The sensitivity of NS1-only RDT for DPO 1 and 2
was 100%, then started to drop from DPO 3 (90.2%) to DPO 5 (75.6%), and through DPO
10 (66.6%) (Figure 2). The sensitivity of NS1-only RDT for different serotypes were
DENV1 (76.8%), DENV2 (89.1%), DENV3 (81%), DENV4 (100%) and co-infections
(100%) (Figure 3A). In the WHO groups A, B and C, the respective sensitivities were 100%,
96.1% and 62.5% (Figure 3B).

NS1 ELISA: The overall sensitivity of the DENV Detect NS1 ELISA was 84.4% with
100% specificity, PPV and NPV was 100% and 53.3%, respectively (Table 1). The
sensitivity of NS1 ELISA in primary and secondary infections was 91.8% and 69.8%,
respectively. The sensitivity of NS1 ELISA for DPO 1 and DPO 2 was 100%, and fell
14
from 92.6% on DPO3 to 75.6% on DPO 5 (with minor variations during the second week
of illness, and finally to 66.6% on DPO 10 (Figure 2). The sensitivity of NS1 ELISA
varied among different serotypes; 81.1% for DENV1, 91.3% for DENV2, 71.4% for
DENV3, and 100% for DENV4 and co-infections (Figure 3A).
The sensitivity of NS1 ELISA in the WHO classified severity groups A, B and C
were 100%, 96.1% and 66.7%, respectively (Figure 3B).

DISCUSSION

NS1/IgM RDT showed overall higher sensitivity in a panel of acute dengue confirmed
samples compared to NS1-only RDT and NS1 ELISA. Sensitivity for NS1/IgM RDT was
high in primary infections, throughout the acute phase of illness, in detecting all the four
serotypes and for different severity levels of dengue infection.

Both the Dengue Day 1 RDT and DENV Detect NS1 ELISA are licensed for use in
India. However this is the first study where these assays are evaluated in the Indian
context by RT-PCR and sequencing using a large cohort of children presenting with
confirmed acute dengue infection, along with a well-tested dengue negative panel. Our
results showed that the RDT showed a higher sensitivity compared to the ELISA test, and
had consistent high sensitivities in detecting primary dengue infections, across clinical
severity groups. In addition, a negative RDT had higher probability of ruling out dengue
infection (NPV 61.2%) compared to ELISA (NPV 53.3%).

A recent study evaluating the NS1 RDT in southern India reported a sensitivity of 91.5%
using a small sample size for the dengue positive panel, but did not address variation
with infection duration, dengue serotypes and clinical severity (Vinodkumar et al., 2015).
The NS1 ELISA has been evaluated in Thailand, the US and Peru and their sensitivities
ranged from 88% to 95% (Anderson et al., 2014; Hermann et al., 2014; Pal et al., 2014).
The NS1 ELISA has been reported to be generally more sensitive than previously
evaluated dengue rapid tests (Hang et al., 2009; Pal, et al. 2014). In contrast, our results
indicate better overall performance by the dengue NS1/IgM RDT. These results suggest
that the NS1/IgM RDT evaluated in this study can be a reliable point-of-care tool for
dengue diagnosis and surveillance in hospital and outbreak settings in India.

15
The Dengue Day 1 RDT (NS1 only and combined) and DENV Detect NS1 ELISA
showed high sensitivity throughout the acute phase of infection, particularly within
the first week.

Generally, dengue NS1 assays show higher sensitivity during early acute phase of illness
and the sensitivity decreases during late acute or convalescent phase of illness
correlating with the kinetics of the NS1 antigen in peripheral blood(Blacksell et al.,
2011; Libraty et al., 2002; McBride 2009). The NS1/IgM RDT showed maximum
sensitivity well into the second week of fever onset, while sensitivity of NS1-only RDT
and NS1 ELISA was reduced. Combining IgM antibody with NS1 antigen in dengue
detection assays prolongs the diagnostic window as IgM antibodies appear later in the
course of illness, and persist longer even after NS1 antigen disappears (Blacksell et al.,
2012; Hunsperger et al., 2014; Wang and Sekaran 2010).

The evaluation panel of acute dengue positive samples used in this study comprised of
all the four serotypes with DENV1 being the most predominant serotype followed by
DENV2, DENV3 and DENV4. Both RDT and ELISA were able to detect all the four
serotypes with varying sensitivities. Overall, the NS1/IgM RDT showed better
performance for all the DENV serotypes with highest sensitivity for DENV2 (95.7%).
However, DENV1, the most frequent serotype in this cohort comprising 53% of all the
serotypes was detected with lower accuracy compared to DENV2 by both the RDT and
ELISA. Several previous studies evaluating NS1 kits have shown higher sensitivity for
DENV1 (Duong et al., 2011; Hang, et al. 2009; Hermann, et al. 2014; Lima Mda et al.,
2011; Osorio et al., 2010). The lower sensitivity for DENV1 seen in this study may be
due to a more divergent strain or a new genotype of DENV1 circulating in this region not
available during the development of the kit. That the NS1/IgM RDT showed high
sensitivity to DENV3 was also observed in a recent study (Hermann, et al. 2014); while
the NS1 ELISA showed decreased sensitivity to DENV3 as shown in other studies
(Blacksell, et al. 2008; Lima Mda, et al. 2011). Both the RDT and ELISA detected
DENV4 and co-infections with 100% sensitivity, although the sample number tested in
these subgroups was small.

16
The sensitivity was higher in primary dengue infections compared to secondary dengue
infections by both the RDTs and ELISA consistent with several other studies. Dengue
Day 1 NS1/IgM RDT showed 95.4% sensitivity in detecting primary infections and
77.3% sensitivity in detecting secondary infections which was higher compared to NS1-
only RDT and NS1 ELISA. The sensitivity among secondary dengue infections are
generally lower than primary infections because of the abundance of immune complex
formation between NS1 antigen and high levels of IgG antibodies in secondary
infections.

The finding that NS1/IgM RDT and ELISA showed increased sensitivity among the mild
dengue group compared to the severe dengue group was reported by other researchers
(Osorio, et al. 2010), although this finding was not consistently reported everywhere
(Guzman et al., 2010; Lapphra et al., 2008). That severe dengue cases are predominantly
secondary infections with high levels of anti-NS1 IgG and low levels of anti-DENV IgM
antibodies explains the lower sensitivity in severe dengue infections(Innis et al., 1989).
However, milder cases are generally primary infections with high levels of anti-DENV
IgM; thus the higher sensitivity. In this study the proportion of secondary dengue
infections among severe dengue cases was 40.2% and 23.5% among milder cases, the
higher proportion of secondary infections explains the lower sensitivity in severe cases
compared to milder cases. However, corroborating with a recent study from India (Singla
et al., 2016) it is also important to note that 50% of severe dengue cases in this study
were primary infections indicating that primary infections can equally cause severe cases.
The combination of NS1/IgM RDT improved detectability by complementing and
compensating for the declining levels of free NS1 in severe dengue cases. The prevalence
of dengue infection was high in the population tested, resulting in a high positive
predictive value of 99%. A negative RDT result, however did not completely rule out
infection with great confidence, as the probability of having dengue infection despite a
negative test was still approximately 39%.

17
This study had some limitations to note; The DENV 3 and DENV 4 samples used in this
study were not large enough to capture the actual performance in these serotypes. This
study enrolled hospitalized patients, therefore a low number of mild dengue cases were
included in this study. Cross-reactivity with other causes of acute febrile illness in
children such as flaviviruses other than dengue, rickettsial and parasitic diseases was not
performed. As this was a single site study performed in a pediatric population admitted to
a tertiary care hospital situated in a dengue-hyperendemic region, the results from this
study need to be interpreted with caution among the adult population and in a community
setting. Nevertheless, this is a large study conducted among patients originating from
several southern Indian states, with well-characterized dengue panels with clinical and
epidemiological classifications, and is well poised to provide fodder for active discourse
and further study into dengue infections, and also to provide important information to
help guide policy on dengue diagnostic testing.

In conclusion our results indicate that the Dengue Day 1 NS1/IgM RDT demonstrates
excellent performance as a dengue diagnostic test. It displays many features that are
desirable in an ideal diagnostic test; it is simple and easy to perform, highly sensitive and
specific, and does not require sophisticated equipment. In addition, its widespread
availability and low cost features make the NS1/IgM RDT a useful tool for rapid and
reliable diagnosis of acute dengue in hospital settings as well as point-of-care settings.

Acknowledgments

We acknowledge the financial support of DBT, Ministry of Science and

Technology,

Government of India (No. BT/PR8470/MED/29/726/2013) for conducting this study. We

thank

our study nurse Mrs. Sheeba and phlebotomist Mr. Prem Kumar for patient recruitment
and

specimen collection. Our sincere thanks to Mrs. Karthika Kumar for data management.
We are

very grateful to our study children and their caregivers for participating in this study.

18
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Highlights

Dengue Day 1 rapid diagnostic test is a simple, point-of-care test for dengue
Exhibits excellent performance for diagnosis of acute dengue infection in children
Shows higher sensitivity compared to NS1 ELISA
Detects all four dengue serotypes

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