The Relationship between Non Structural 1 (NS1) Antigen Test

Result to Symptoms, Clinical Signs and Platelets Count on Patients

With Suspected Dengue Infection in Urip Sumoharjo Hospital

Abstract
Background: The diagnosis of dengue infection early on is very important. Currently it has developed an
antigen non-structural 1 (NS1) examination of the dengue that can detect infection with the dengue virus
early. However, not all health care centrers have adequate laboratory facilities for inspection NS1 dengue.
Platelet count and clinical manifestasions are indicators wich become the basis of a diagnosis in health
centers with limited facilities.
Objective : To determine the relationship between the NS1 antigen test result to symptoms, clinical signs
and platelets count in patients with suspected dengue infection.
Methods : This study uses a observasional analytic method with cross sectional approach. Research
conducted at the Clinical Pathology Laboratory and Inpatient Room of Urip Sumoharjo Hospital in October-
November 2016. Total sample of 30 people is determined. NS1 antigen test performed using
immunochromatography rapid test. Platelet examination performed using hemanalizer. Clinical signs and
symptoms obtained from medical records records of patients.
Results : Fisher's exact test of the relationship between the results of NS1 to platelets count obtained p
value of 0.031, meanwhile the relationship between NS1 antigen test results against clinical signs and
symptoms obtained p value of 0.115.
Conclusions : There is a significant correlation between the results of NS1 antigen test against platelet
count, but there is no correlation between the results of NS1 against clinical signs and symptoms in patients
with dengue.

Keywords: Dengue, NS1, platelet

Korespondensi: Muhamad Jyuldi Prayoga, S.Ked, alamat Jl. Bumimanti 4, Kampungbaru,

Bandarlampung, HP 083160599172, e-mail jyuldip@yahoo.com

INTRODUCTION pain, which is accompanied by

Dengue infection is currently
one of the major health problems in the
world. Over the past three decades,
there has been a considerable global
increase in the frequency and epidemic
of dengue fever (DF), dengue
haemorrhagic fever (DHF), and dengue
shock syndrome (DSS) as the incidence
of the disease increases. In the period
2000 to 2008 the average number of
cases per year was 1,656,870 cases, the
number increased by almost three and
a half times compared to the number of
cases in the period 1990-1999 which
amounted to 479,848 cases.1
Dengue fever (DF) and dengue
haemorrhagic fever (DHF) are infectious
diseases caused by the dengue virus
which is transmitted through the
mosquitoes Aedes aegypti and Aedes
albopictus. This disease is characterized
by fever, muscle pain and / or joint
leukopenia, rash, lymphadenopathy,
thrombocytopenia and hemorrhagic
diathesis. 2,3
DHF cases in Lampung Province
during 2004-2014 tended to fluctuate.
The number of dengue morbidity in
Lampung Province in 2014 amounted to
168 per 100,000 population (above
National IR which is 51 per 100,000
population). The number was a
decrease from the previous year, which
was 58.08 per 100,000 population.
However, there was an increase in case
fatality rate (CFR) in the year from
0.98% in 2013 to 1.63% in 2014.4 The
increase in CFR can occur due to various
factors, one of which is inadequate
patient handling.
Dengue fever can develop very
quickly in a few days, even in a matter
of hours the patient can enter in a
critical state.5 Shock and organ
disturbances have been proven to be
the main factors causing death in
dengue infection.6,7,8 Thrombocytopenia
and plasma leakage are the causes
shock to DHF. Patients with bleeding
manifestations have a mortality ratio 3-
4 times greater than patients without
bleeding manifestations.
Thrombocytopenia is an important
parameter in handling DHF patients.
Thrombocytopenia or platelet
deficiency, is a condition in which
platelets in the circulatory system are
below normal (150,000- 450,000 / μl of
blood) .9,10
The diagnosis of dengue
infection as early as possible is very
important to prevent the progression of
the severity of the disease into a more
serious form. But the diagnosis of
dengue is difficult to enforce on the first
few days of illness because the
symptoms that appear are not specific
and difficult to distinguish from other
infectious diseases so that it can cause a
delay in diagnosis. Diagnosis of dengue
disease in addition to assessing clinical
symptoms also requires laboratory
examinations to help diagnose.
Laboratory diagnosis of dengue
infection can be established by
detecting specific viruses, genome
sequences, antibodies, and viral
antigens.11 There is now an examination
of nonstructural antigen 1 (NS1) that
can detect or diagnose dengue virus
infection earlier, even on the first day of
fever onset because NS1 protein
circulates in high concentrations in the
patient's blood during the initial acute
phase. NS1 is an abundant glycoprotein
produced by viruses during the early
stages of infection found in infected
cells in cell membranes and secreted
into extracellular space.11,12
NS1 examination is very useful
because supportive therapy and patient
monitoring can be carried out
immediately so that it can reduce the
risk of complications and death.13
However, not all health care centers,
especially in peripheral areas, have
adequate laboratory facilities for
examining dengue specific markers such
as NS1 . Platelet counts are the only
simple investigations that can be
performed in the peripheral area with a
calculation using a microscope.
Based on the above background, in this
study the researcher intends to find out
that there is no correlation between
NS1 examination results of symptoms,
clinical signs and platelet counts in
patients suspected of dengue infection.
Research purposes
Knowing whether there is a
relationship between the results of NS1
antigen examination of symptoms,
clinical signs of dengue infection, and
platelet counts in patients suspected of
dengue infection.
METHODS
Design
This type of research is an observational
analytic study with a cross sectional
approach.
Place and time
The study was conducted in
May-December 2016 in the Clinical
Pathology Laboratory and inpatient care
at Urip Sumoharjo Hospital in
Bandarlampung.
Research Subjects
Population
Affordable population for this
study were patients suspected of
dengue infection in Urip Sumoharjo
Hospital Bandarlampung in October-
November 2016.
Sample
The sample used in this study is
patients suspected of dengue infection
in Urip Sumoharjo Hospital
Bandarlampung in October-November
2016 with the criteria for duration of
illness at admission to 1-5 days from the
onset of fever. The sampling technique antigen test device. The platelet count
in this study was consecutive sampling. data was obtained from the results of a
based on calculations, the number of complete blood examination in the
samples in this study were 30 people. blood sample of patients who were
examined using a hematology analyzer.
Sample Criteria Secondary data in this study are
Exclusion criteria symptoms and clinical signs in patients
1. Patients suspected of dengue suspected of dengue infection.
with prolonged illness since the Secondary data was obtained through
onset of fever is more than 5 identification and recording of patient
days. status / medical records, and interviews
2. Patients who are taking drugs with nurses in the inpatient room of
that suppress bone marrow. Urip Sumoharjo Hospital. Data
3. Patients who have a history of obtained, then carried out univariate
blood disorders. analysis and bivariate analysis using a
4. Patients with other co-infected computer statistics program.
diseases, such as typhoid fever.
5. Medical records are Univariate Analysis
incomplete. a. Overview of NS1 Antigen
6. Patients with symptoms and Examination in Suspected Patients
signs of shock. with Dengue Infection
The results of NS1 antigen
Research variable examination in patients suspected of
The independent variable in this dengue infection are described in
study was the result of nonstructural the following table:
antigen examination 1 (NS1). The Table 1. NS1 examination results
dependent variable in this study is the NS1 Frequency Percent
clinical symptoms of dengue infection Negative 10 33,3
Positive 20 66,7
and platelet count. The clinical Total 30 100
symptoms referred to in this study were
fever, which was accompanied by at
least 2 of the following symptoms: b. Overview of Platelet Counts in
headache, retroorbital pain, myalgia, Suspected Dengue Infection
arthralgia, rash, and bleeding Patients
manifestations of headache, Based on the results of the
retroorbital pain, myalgia, arthralgia, study, the description of platelet
rash, and bleeding manifestations such counts in patients suspected of
as petechiae, test for positive dengue infection can be seen in the
tourniquet, and spontaneous bleeding following table:
Table 2. Overview of Platelet Counts in
RESULTS AND DISCUSSION Suspected Dengue Infection Patients
Results
Primary data was obtained by Platelet Frequency Percent
countss
conducting NS1 antigen examination
>150.000/mm3 5 16,7
and platelet count at the Clinical
Pathology Laboratory at Urip Sumoharjo <150.000/mm3 25 83,3
Hospital. NS1 antigen examination was
Total 30 100
carried out using the rapid
immunochromatography-test method
using the SD Bioline ™ dengue NS1
c. Symptoms and Clinical Signs in 4 2
Suspected Patients with Dengue 5 8
Total 30
Infection Mean 3,23
Based on the results of the Median 3
study, the symptoms and clinical
signs in patients suspected of Bivariate Analysis
dengue infection are described in The data obtained by the
the following table: researchers were analyzed
bivariately using an alternative test
Table 3. Symptoms and clinical signs of from the chi square test, which was
dengue infection
a test of fi ners exact. The fisher's
Symptoms and Frequency Percent
clinical signs of exact test is used because the data
dengue infection obtained does not meet the chi
9 30 square test requirement, which is
<2 Symptoms
and clinical signs the expected count that is less than
of dengue 5 by> 20%.
infection
21 70 a. Relationship between NS1
≥2 gejala/tanda
klinis infeksi Antigen Examination Results
dengue and Platelet Counts
Total 30 100
The results of the bivariate
analysis of the relationship
d. Degree of Disease Severity in between the results of NS1
Samples antigen examination and platelet
Based on the results of the count can be seen in the
study, the severity of dengue disease following table:
in the sample can be seen in the
following table: Table 6. Relationship between NS1
Table 4. Degree of Disease Severity Antigen Examination Results and
Degree of Frequency Percent Platelet Counts
Disease NS1
Severity To
DF 7 23,3 Posi Nega
tal p
DHF grade I 20 66,7 tive tive
valu
DHF grade II 3 10 e
DHF grade III 0 0 <150.000/ 19 6 25
DHF grade 0 0 Platelet mm3 0,03
IV counts >150.000/ 1 4 5 1
Total 30 100 mm3
20 10 30
Total
e. Duration of Fever in Samples
The old characteristics of fever in the
sample can be illustrated in the b. Relationship of NS1 Antigen
following table: Examination with Symptoms
and Clinical Signs of Dengue
Tabel 5. Lama demam pada sampel Infection

Day-to Frequency The results of the bivariate

day fever analysis of the relationship
1 1
between the results of NS1
2 9
3 10 antigen examination with
 symptoms and clinical signs of
dengue infection can be seen in
the following table:
Table 7. Hubungan hasil pemeriksaan
antigen NS1 terhadap gejala
dan tanda klinis infeksi dengue
NS1
Total
Positiv Negati
e ve
≥2 16 5 21
Sympto sympt
ms and oms/
clinical signs
signs of <2 4 5
dengue sympp
toms/s
igns
20 10
Total
Discussion
Based on the results of the
study, of the 30 samples tested there
were 20 samples with positive NS1
examination results and 10 other
negative samples. The percentage of
thrombocytopenia that occurred in all
tested samples was quite high, which
was 83.3%, while only 16.7% of the
samples had normal platelet counts (>
150,000 / mm3). The researcher used
WHO diagnosis criteria (2011) in
determining the symptoms and clinical
signs of dengue infection, namely fever
accompanied by 2 or more symptoms /
signs: headache, retroorbital pain,
myalgia, arthralgia, rash, bleeding
manifestations. Table 5 shows, 21
samples met the diagnosis criteria for
symptoms and clinical signs of dengue
infection and 9 other samples had non-
specific symptoms with only <2
symptoms / clinical signs.
Based on Fisher's exact test
regarding the relationship between the
results of NS1 antigen examination of
platelet count, which is presented in
table 8, the results of p value are 0.031.
The value of the p value obtained is
smaller than the significance value or α
(α = 0.05). This means that Ho is
rejected or it can be concluded that
there is a significant relationship
between the results of NS1 antigen
examination of platelet counts in
patients suspected of dengue infection.
Meanwhile, the results of the fisher
exact test on the relationship between
p value the results of NS1 antigen examination
of the symptoms and clinical signs of
0,115 dengue infection, presented in table 9,
obtained a p value of 0.115. The value
9 of p value obtained is greater than the
value of α (α = 0.05), which means that
Ho is accepted. It can be concluded that
30 there is no relationship between the
results of NS1 antigen examination with
symptoms and clinical signs of dengue
infection.
The results of this study are in
accordance with the results of a
previous study conducted by Badave et
al (2015) 13 that there is a significant
relationship between the results of NS1
antigen examination on the number of
platelets and the chi square test
obtained p value which is equal to
0,0001. Other studies by Santosh et al
(2013) and Kulkarni et al (2011) also
showed a significant relationship.14,15 In
the Santosh et al study, of 56 cases of
NS1 positive, 50 cases or 89.28% of
cases occurred thrombocytopenia.
Meanwhile, a study conducted by
Kulkarni et al. Aimed to determine the
relationship of thrombocytopenia with
serological examination and dengue
NS1 antigen. The results of the Kulkarni
et al study state that the correlation
between NS1 examination results is
quite significant, with a p value of
<0.001. However, after further analysis,
it was found that the results of NS1
antigen examination coupled with
serological examination (IgM-anti
dengue) had a more significant
relationship to the occurrence of
thrombocytopenia compared to NS1
antigen examination alone.14,1
 The diagnosis of dengue disease
in addition to routine clinical and
hematological assessment also requires
a laboratory examination to detect
viruses. Currently laboratory tests to
diagnose dengue virus have developed
rapidly so that sensitivity and specificity
are better with faster examination
times. NS1 antigen examination is one
of the examinations that can detect
dengue virus infection earlier even on
the first day of fever onset.16 NS1
antigen in dengue virus is a 46-50
kilodalton glycoprotein expressed on
infected host cells both membrane
associated (mNS1 ) and secreted (sNS1)
and not part of the structural
component of virion. 12 NS1 is
produced by all flaviviruses and plays an
important role in the process of
replication and survival of the virus. 17
During dengue infection,
dengue virus can induce the occurrence
of clinical manifestations with a varied
spectrum. Dengue infection usually
causes thrombocytopenia in both mild
and severe forms.18 Thrombocytopenia
is one of the criteria used by WHO as an
indicator for diagnosis and knowing the
clinical severity of dengue infection.
Based on WHO diagnosis guidelines
(2011), indicators of the diagnosis of
thrombocytopenia in dengue infection
are described as platelet counts
<150,000 per microliter of blood. Based
on research, platelet counts decreased
significantly on day 4 of fever.1,19
NS1 antigen is an important
immunogen in the pathogenesis of
dengue infection and plays a role in the
occurrence of plasma leakage and
bleeding in dengue infection. The
results of the study conducted by Duyen
et al (2011) stated that the high level of
NS1 antigen on day 3 of the infection
was associated with a decrease in
platelet count.20 There are various kinds
of NS1 antigen mechanisms in inducing
thrombocytopenia, including
autoimmune platelet activation and
destruction , platelet dysfunction, and
direct infection.18
NS1 antigens induce platelet
lysis mediated by activation of the
complement system which causes a
decrease in platelet count in the
circulation. In addition, there is an
autoantibody reaction with the initial
NS1 target that attacks platelets and
fibrinogen.21 The process of
autoantibodies is due to the molecular
mimicry mechanism because the C-
terminal part of NS1 shows a sequence
that is homologous with integrins on
the platelet surface. Similarly, in other
parts, namely capsid, M protein, and
protein E have homologous sequences
with coagulation molecules such as
thrombin, plasminogen and tissue
plasminogen activator.22 Some
evidence indicates that antibodies to
NS1 proteins can also cross-link with
platelets and endothelial cells, which
can subsequently induce platelet
destruction and endothelial damage in
patients with dengue infection. 23
Another study by Rachman et al
(2013) and Cheng et al (2009) reported
that anti-NS1 antibodies also cause
platelet dysfunction. Anti-NS1
antibodies will bind and inhibit the
activity of Protein Disulfide Isomerase
(PDI) so that it causes interference with
platelet aggregation induced by ADP.
The binding of anti-NS1 antibodies with
PDI is caused by the presence of
homologous sequences between 311-
330 amino acid residues in DENV NS1
with PDI domain, thioredoxin. Dengue
NS1 antigen can also bind and inhibit
prothrombin activation, which
contributes to the slowdown of partial
thromboplastin time (APTT) activation
and bleeding in dengue patients.24,2
In this study also found that
there was no relationship between the
results of NS1 antigen examination of
symptoms and clinical signs of dengue
infection. In table 7 shows there are 16
people with positive NS1 results with ≥2
symptoms and clinical signs of dengue et al (2011) reported that there are
infection and 4 NS1 people positive but differences in the number of NS1
the symptoms of dengue infection are proteins secreted based on a strain of
not specific (<2 symptoms / signs). In virus that infects the host. The sNS1
patients with negative NS1 examination concentration is much higher in DENV-1
results, there were 5 people with infections than in DENV-2. The high
positive clinical symptoms of dengue concentration of sNS1 and the level of
(≥2 symptoms / signs), and 5 patients viremia can lead to thrombocytopenia,
with other negative NS1 had negative hemoconcentration and more severe
dengue symptoms. These results can be symptoms
influenced by several factors, including: This type of infection also
duration of fever or onset of infection, affects the clinical symptoms that
infectious virus strains, type of infection appear. The results of research
(primary or secondary), age, and sex. conducted by Pusparini (2004) Clinical
The results of research symptoms in primary and secondary
conducted by Ahmed and Shobha dengue infections are almost the same
(2014) showed that the sensitivity and but in dengue infections secondary to
specificity of NS1 antigen examination bleeding symptoms are more
28
had differences based on the patient's common. In addition, clinical
fever. On the first day of fever, the symptoms that appear are influenced
sensitivity of NS1 examination is not so by the age of the patient. Based on
high, which is equal to 50%. Day 2 of research conducted by Kittigul et al
the fever showed the highest sensitivity (2007), the clinical manifestations of
level of 100%. Furthermore, it declined dengue infection appear more
on day 3 to 71.4%, and 75% on day 4. frequently in adults than children.
Specificity of NS1 examination showed Clinical manifestations include
quite good results with 100% specificity. petechiae, melena, headache,
26 Meanwhile, in this study, the mean retroorbital pain, arthralgia, myalgia,
and median duration of fever were nausea, and vomiting. Changes in
3.323 and 3. In 5 negative NS1 samples laboratory examinations such a also
who had positive dengue symptoms 2 higher in adults than in children.29
of them came with day 2 fever, 1
person on day 3 of fever and 2 other CONCLUSION
people on day 5 of fever, and if the Based on the results of this study, the
mean is taken at 4.25 days. Based on conclusions are as follows:
the results of this study, there were also 1. There is no significant
4 samples with positive NS1 with relationship between the
negative dengue symptoms. In the 4 results of NS1 antigen
samples, 2 samples came on day 2 of examination of symptoms and
fever, and 2 others on day 3 of fever. In clinical signs of dengue
the early days of dengue infection infection in patients suspected
clinical manifestations may not be of dengue infection.
specific. Manifestations of bleeding and 2. There is a significant
thrombocytopenia in dengue generally relationship between the
appear after the 3rd day of fever.27 results of NS1 antigen
The sensitivity of the NS1 examination of platelet counts
antigen examination can also be in patients suspected of dengue
affected by the level of viremia and the infection.
number of NS1 secreted by the virus. 3. The percentage of positive NS1
Based on research conducted by Duyen results in patients suspected of
 dengue infection in Urip
Sumoharjo Hospital is 66.7%.
4. The percentage of
thrombocytopenia in patients
suspected of dengue infection
in Urip Sumoharjo Hospital is
83.3%.
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