Chaudhary et al., 2016. Journal of Genes and Cells, 2(1): p, 11-15 www.imaqpress.

com
doi: 10.15562/gnc.32

NS1: An early Diagnostic Tool for Dengue Virus Fever

Affifa Kiran Chaudhary.1,2*, Mehwish Anwer1, Mahmood Hussain Qazi1*
1. Centre for Research in Molecular Medicine (CRiMM), The University of Lahore, Lahore, Pakistan
2. Department of Chemical Engineering, UAE University, Al-Ain, Abu Dhabi, UAE

ABSTRACT

Dengue virus is a highly prevailing unremitting pathological menace in the developing countries like Pakistan.
The variation in the type and frequency of dengue infection demands a continuous surveillance on its spread and
diagnosis, in order to develop appropriate management and therapeutic strategies. Dengue Virus is highly complex
disease with various manifestations. It is hard to characterize the dengue viral infection with ordinary laboratory
tests. However, NS1 could serve a good diagnostic tool in the first few days of fever. In this research, 88 patients
from Lahore regions of Punjab were analysed for the presence of dengue specific NS1 antigen from 1 to 30 days
of dengue fever. ELISA based kit was employed. The obtained data was analysed expressed percentage frequency
of NS1 positive and negative along with IgM positive and negative in all dengue fever patients. Importantly it was
observed that 36.3 % were infected by dengue. NS1 antigen was efficiently quantified at earlier days of infection.
IgM was negative in all the subjects who were positive for NS1 on the second day of fever. However, this
association weakened with the progression of fever. It was analysed that thrombocytopenia and leuckocytopenia
are not linked with the NS1 positivity and these states were random. It can be inferred that dengue diagnosis is
very complicated and the antigenic profile dramatically changes with the progression of fever in terms of days.
Clinicians should employ a diagnostic method based on a comprehensive analysis of subjects in order to minimize
the risk of dengue shock syndrome resulting in haemorhage.
Keywords: Dengue virus fever, NS1, dengue outbreak in Pakistan, Viral borne diseases in Pakistan

Dengue is a widespread viral borne infectious antibody dependent enhancement (ADE) infection of
fever in humans of the tropics and subtropics of the peripheral leukocytes. It is also hypothesised that
world (1). It has been estimated that near about 100 there is an association between dengue viremia illness
million cases, 500,000 cases of dengue and disease outcomes in patients with secondary
haemorrhagic fever (DHF) and 12000 deaths occur dengue virus 1 and dengue virus 2 infections but there
worldwide annually due to of dengue fever (2). was no any association found in patients with primary
DENV has become the most important arthropod- dengue virus 1 infection. The severity of diseases is
borne virus affecting human (3). directly related to the dengue virus 3 viremia
In dengue infection, viremia usually hits the peak (measured as dengue virus 3 genome equivalent
at the time or shortly after the onset of illness and levels) and subsequent immune activation. There is
can be detected within 2 to 12 days of illnes. The also direct relationship between the magnitude of
number of viral infected cells can determines the viremia and the magnitude of plasma leakage.
severity of illness and is directly related to Sensitive and reproducible quantitative RT-PCR
* Correspondence:
Email: dr.affifa@uaeu.ac.ae Received 11-11-2015 Accepted 18-12-2015
____________________ Published 08-02-2016
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Chaudhary et al., 2016. Journal of Genes and Cells, 2(1): p, 11-15 www.imaqpress.com
doi: 10.15562/gnc.32

assays have been reliably used to find out the MATERIAL AND METHODS
severity of disease and viremia level (4, 5).
Flavivirus are single-stranded, positive-sense Sample Collection
RNA enveloped viruses. Their genomic RNA is During 2011 dengue epidemic in Lahore, Punjab,
about 11 kb long and contains 10 genes encoding Pakistan, blood samples of 88 children who presented
three structural proteins namely, capsid [C], with fever and were suspected of being infected with
envelope [E], and membrane [M]) and seven non- dengue virus infection; were collected for the
structural proteins (NS1, NS2a, NS2b, NS3, NS4a, descriptive, prospective and retrospective
NS4b, and NS5) (6). The polycistronic coding questionnaire based study. Samples were collected
region is flanked by non-coding regions at its 59 from Dengue Ward, Children Hospital, Ferozpur
and 39 ends. The non-structural protein, NS1, is a Road, Lahore. Detailed physical examination was
highly conserved glycoprotein, but its biological performed and all the available blood test reports
activity has not been established. During in vitro were taken. Questionnaires were duly filled with bio
infection, the flavivirus NS1 protein is expressed data of the child, clinical presentation of the illness,
as an intracellular membrane-associated form Complete Blood Count (CBC) record, along with
essential for viral replication (7) or as a cell available additional investigative information.
surface-associated form that may be involved in Children with diseases like diabetes, cirrhosis, CVD
signal transduction (8). The patients with DHF or kidney disease were not included in the study.
were found to have high levels of NS1 protein in
their serum, therefore NS1 could be useful Biochemical Analysis
prognostic indicator in DHF. All the biochemical analysis were performed at the
In solution, secreted NS1 protein behaves as a Centre for Research in Molecular Medicine
hexamer; it circulates and accumulates in the sera (CRIMM), The University of Lahore. Serum was
of dengue virus-infected patients throughout the centrifuged at 4000 rpm for 10 minutes and then
clinical phase of the disease (9, 10). A recent study aliquoted and preserved at -20 C. Serum
demonstrated that soluble NS1 protein binds to concentration of NS1 antigen was measured by using
endothelial cells and are recognized by anti-NS1 commercial ELISA kits by BioRad (according to
antibodies which could contribute to plasma manufacturer protocol).
leakage as the dengue virus infection severe more
(11). The detection of secreted NS1 protein Statistical Analysis:
represents a new approach to detect acute dengue The significance of difference between quantitative
infection. A commercially developed diagnostic variables was analysed by 2-tailed Students t-test.
test based on dengue NS1 antigen-capture ELISA Pearson test was used to calculate correlation
was investigated in two studies (one in South between variables of interest. P value < 0.05 was
America with an overall sensitivity of 88.7% and considered statistically significant. All calculations
the other one in Southeast Asia with an overall were carried out with the SPSS version 19 (SPSS, Inc,
sensitivity of 93.4% and both of these test showed Chicago, IL, USA).
100% specificity (12).The present research work is
to evaluate the NS1 antigen in dengue infected
patients and the level of viremia with dengue
disease severity using well characterized sera from
the patients presenting at hospitals.

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doi: 10.15562/gnc.32

Table 1:
Expression Pattern of NS1 and IgM in 80 Dengue Virus Fever Patients at Different Days (1 to 30)

Days of Total NS1 %age IgM % of NS1 %age IgM - % Equivocal

fever %age +ve of +ve IgM+ -ve ve %
NS1+
1 5 5.6 1 20.00 1 20.00 4 80.00 4 80 - -
2 7 7.9 5 71.43 0 0.00 2 28.57 7 100 - -
3 15 17 7 46.67 8 53.33 8 53.33 7 46.6667 - -
4 12 13.6 3 25.00 11 91.67 8 66.67 1 8.33333 1 8.33
5 14 16 4 28.57 12 85.71 10 71.43 2 14.2857 - -
6 8 9 3 37.50 7 87.50 4 50.00 1 12.5 1 12.50
7 12 13.6 5 41.67 8 66.67 7 58.33 4 33.3333 - -
8 3 3.4 1 33.33 1 33.33 1 33.33 2 66.6667 1 33.33
10 2 2.2 - - 1 50.00 2 100.00 1 50 - -
11 1 1.1 - - 1 100.00 1 100.00 0 0 - -
12 3 3.4 - - 3 100.00 3 100.00 0 0 - -
14 2 2.2 1 50.00 1 50.00 - - 1 50 1 50.00
20 2 2.2 1 50.00 2 100.00 1 50.00 0 0 - -
30 2 2.2 1 50.00 2 100.00 1 50.00 0 0 - -

RESULTS DISCUSSION

Total number of patients included in the study was Provision of proper treatments to the patient depends
88. Frequency of male subjects was 47 making largely upon an early laboratory diagnosis of acute
53.5% of total whereas; female subjects were dengue virus infection. The patient may die of
45.5% of total with representative frequency of 41. complications within first 24 hours if this integral
The male to female ratio came out to be 1.17:1. component is overlooked. Currently various methods
The subjects which were analyzed on the basis of are available for the diagnosis of severity of the
NS1 antigen were descriptively analyzed based on disease. However, there is a severe deficiency of
their day of fever. It was observed that NS1 was complete data about the diagnosis and pathogenesis
detected in 71.43% of the total subjects on 2nd day of the virus in association with period of illness.
of fever. However this efficiency decreased with Moreover, little information is in hand about
increasing days and more number of samples were antigenic presentation of dengue fever in children of
diagnosed negative by NS1 ELISA. It was also the Pakistani population.
observed that on day 2 IgM and NS1 were 100% In the present study, children suffering from fever
correlated. were suspected of dengue virus infection. They were
tested with dengue specific NS1 ELISA kits for
diagnosis of dengue.

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doi: 10.15562/gnc.32

Previous studies have reported sensitivity greater Also, IgM correlated well with NS1 in early days but
than 90% for diagnosis of dengue fever. Based on later the association was poor.
the test, the subjects were then classified into
dengue positive and dengue negative subjects. CONCLUSION
Based on WHOs classification of three phases i.e.
acute phase (2-7 days after onset of fever), afebrile Dengue virus is highly complex disease with various
phase (8-11 days after fever onset) and manifestations. It is hard to characterize the disease
convalescent phase (18 days onwards), among 73 with various tests. However, NS1 could serves a good
patients in the first phase, 28 were dengue positive diagnostic tool in the first few days. It was also
with 76.71% of total and 43 individuals were concluded from the current study that
dengue negative with 58.0% of total. All the thrombocytopenia and leucocytopenia is because of
subjects found positive and negative for NS1 the serotype and is not particularly associated with
antigen were reassessed with respect to the day of any antigen and antibody.
presentation of fever. It was observed that NS1
was clearly detected in early febrile phase and the
ability decreased with increasing day of fever.

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doi: 10.15562/gnc.32

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