|Year : 2015 | Volume
| Issue : 2 | Page : 95-99
Early dengue diagnosis: Role of rapid NS1 antigen, NS1 early ELISA, and PCR assay
Vaishali N Solanke, Mohan G Karmarkar, Preeti R Mehta
Department of Microbiology, Seth Gordhandas Sunderdas Medical College and King Edward Memorial Hospital, Mumbai, Maharashtra, India
|Date of Web Publication||9-Jun-2015|
Dr. Vaishali N Solanke
Department of Microbiology, 5th Floor, MSB Seth Gordhandas Sunderdas Medical College and King Edward Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra
Context: Early and accurate dengue diagnosis is important for disease surveillance, and also to start effective control measures in endemic countries. Aims: In this study, we evaluated the performance of three techniques available for early dengue diagnosis, viz., rapid nonstructural 1 (NS1) antigen test, NS1 early enzyme-linked immunosorbent assay (ELISA) test, and reverse transcription polymerase chain reaction (RT-PCR) test. Settings and Design: Retrospective analytical study. Materials and Methods: Performance of three different techniques, viz., rapid NS1 antigen, NS1 early ELISA, and RT-PCR on serum samples of suspected dengue cases. Statistical Analysis Used: Sensitivity, specificity, negative predictive value, and positive predictive value calculated for rapid test and ELISA test, keeping PCR as the gold standard Results: Amongst 155 samples received from clinically-suspected dengue cases within 1-9 days of fever, 56 samples were received during 1-3 days, 52 samples were received during 4-6 days, and 47 samples were received during 7-9 days. Of these 155 samples, 38 (24.5%) were positive by rapid NS1 antigen, 47 (30.3%) by NS1 ELISA, and 54 (34.8%) by reverse transcriptase polymerase chain reaction (RT-PCR). Rapid NS1 antigen and NS1 ELISA showed the highest positivity on days 1-3, while highest positivity for PCR was on days 4-6. The sensitivity and specificity of NS1 ELISA were 66.6% and 89.1%, while sensitivity and specificity of rapid NS1 antigen were 55.5% and 92%, respectively. Conclusions: With high mortality and morbidity associated with dengue, it is imperative to diagnose the disease during the early phase. All three assay formats are helpful in early diagnosis and to provide information of dengue cases in time.
Keywords: Dengue, early, enzyme-linked immunosorbent assay, polymerase chain reaction, rapid
|How to cite this article:|
Solanke VN, Karmarkar MG, Mehta PR. Early dengue diagnosis: Role of rapid NS1 antigen, NS1 early ELISA, and PCR assay. Trop J Med Res 2015;18:95-9
|How to cite this URL:|
Solanke VN, Karmarkar MG, Mehta PR. Early dengue diagnosis: Role of rapid NS1 antigen, NS1 early ELISA, and PCR assay. Trop J Med Res [serial online] 2015 [cited 2020 Feb 25];18:95-9. Available from: http://www.tjmrjournal.org/text.asp?2015/18/2/95/158402
| Introduction|| |
Dengue virus (DENV) infection is one of the most important arthropod-borne viral diseases in tropical and subtropical countries throughout the world. 
DENV causes various clinical symptoms ranging from asymptomatic or undifferentiated fever, known as dengue fever (DF), to fever with plasma leakage, called dengue hemorrhagic fever (DHF). Some cases of DHF become an even more serious form, called dengue shock syndrome (DSS), leading to death, especially among children. 
The DENV 1-4 belongs to the genus Flavivirus of the Flaviviridae family, is transmitted by Aedes aegypti. The genome of single strand RNA virus is composed of three structural protein genes that encode the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven nonstructural (NS) protein genes: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5.  Among the NS proteins, NS1 is a highly conserved glycoprotein, which is essential for virus replication. During the acute phase of DENV infection, NS1 protein is found associated with intracellular organelles and can be transported via cellular secretion pathway to the infected cell surface. NS1 protein was also released from infected mammalian cells and may then be found circulating in the sera of patients. 
Laboratory confirmation of dengue relies on demonstration of the presence of DENV by (a) Isolation of DENV from patient serum, (b) detection of viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR), and (c) detection of dengue specific antibodies by using enzyme-linked immunosorbent assay (ELISA). 
Each of these tests has drawbacks. The usage of dengue-specific antibody detection assays, though cost-effective, is limited in the early phase of the disease as antibodies become detectable only around the fifth day upon the onset of the disease.  Moreover, a single serological detection of immunoglobulin (IgM) is merely indicative of a recent DENV infection, and should not be interpreted as a confirmatory diagnosis of acute infection without a paired second serum sample.
Viral isolation by cell culture and subsequent detection by immunofluorescence, though a gold standard,  cannot be used as a routine diagnostic procedure due to its low sensitivity, laborious procedure, and time consumption. In case of molecular methods, the requirement of a highly trained staff, the need of sophisticated equipment as well the cost factor has limited its application as a routine diagnostic assay. 
Currently, rapid antigen and antigen-capture ELISA commercial kits for detection of NS1 antigen have been developed and evaluated. Detection of dengue NS1 antigen represents a new approach for the diagnosis of acute dengue infection.  In this study, we evaluated rapid NS1 antigen, NS1 ELISA, and polymerase chain reaction (PCR) for early dengue diagnosis in acute febrile illness.
| Materials and Methods|| |
This study is the retrospective analysis of 155 samples obtained during monsoon season, over a period of 4 months from June 2013 to September 2013. Patients of all age groups, clinically suspected of dengue, presented within 1-9 days of fever were included in the study, which was carried out in a tertiary care teaching hospital in Mumbai. The serum was separated and subjected to early dengue diagnostic tests, viz., rapid NS1 antigen, NS1 early ELISA, and PCR. The duration of fever (in days) and other relevant clinical information were recorded from the requisition form.
Dengue rapid NS1 antigen test
The assay was performed in accordance with the manufacturer's instructions (Aspen Laboratories, Ib. International, Inc. Seattle, USA).The dengue NS1 antigen rapid test is an in vitro immunochromatographic test (ICT), one-step assay designed for the qualitative determination of DENV NS1 antigen in human serum and plasma for the diagnosis of early acute dengue infection. About 50 μL of patient serum was added to the sample well marked "S" and three drops of buffer were added in the buffer well marked "B". The test result was interpreted in 15~30 min. The presence of only one color line within the result window indicated negative result and the presence of two color lines ("T" band and "C" line) indicated a positive result. When no control line (C) was found, the test was concluded as invalid.
Dengue NS1 early ELISA test
The assay was performed in accordance with the manufacturer's instructions (Panbio, Brisbane, Australia). Briefly, 100 μL of diluted (1:10 in serum diluent) patient serum, positive control, negative control, and calibrator were added to microwells precoated with a polyclonal capture anti-NS1 antibody and then incubated for 1 h at 37°C. The plates were washed six times and incubated for an additional 1 h at 37°C following the addition of horseradish peroxidase (HRP)-conjugated anti-NS1 monoclonal antibody (MAb). After an additional six washes, antibody complexes were detected by adding tetramethylbenzidine (TMB) and incubating samples for 10 min at room temperature. The reaction was stopped by adding stop solution (1M H 3 PO 4 ), and the plates were read. The cutoff value was determined by multiplying the average optical density (OD) of the calibrator (tested in triplicate) by the lot-specific calibration factor (provided in the kit insert). An index value was calculated by dividing the average OD of each sample by the cutoff value. Index values of <0.9, 0.9-1.1, and >1.1 were considered negative, equivocal, and positive, respectively.
Dengue PCR detection
Dengue genomic RNA was detected by real time RT-PCR method by the TaqMan® RNA Amplification Kit (Roche Molecular Systems, Inc.). This molecular test is considered as the gold standard for early diagnosis and was carried out at the reference laboratory for PCR testing for Municipal Corporation of Greater Mumbai (MCGM) hospitals.
Diagnostic accuracy of rapid NS1 antigen, NS1 early ELISA, with PCR were evaluated. The results were calculated using diagnostic tests that considered sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) as follows: Sensitivity: a/a + c × 100%; specificity: d/d + b × 100%; negative predicted value: d/d + c × 100%; and positive predicted value: a/a + b × 100%, where: a = number of true positives, b = number of false positives, c = number of false negatives, and d = number of true negatives. The rapid NS1 antigen and NS1 early ELISA were compared with RT-PCR.
There is no conflict of interest between the suppliers and producers of the kits and the public health laboratories. We have analyzed only information retrieved from the laboratory databases. All samples included in the study were received by the laboratory for dengue diagnosis. The identities of the patients were kept confidential and biological samples were identified only by numbers.
| Results|| |
In the present study, male to female ratio was 1:1.1, and the age group most commonly affected was 0-20 years, with 81.9% (127/155) samples belonging to this group [Figure 1].
Percentage of positive results by rapid, ELISA, and PCR methods with duration of fever
The serums were divided into three groups with regard to duration of fever (1-3, 4-6, and 7-9 days). The maximum number of 56 samples was received during 1-3 days of fever, followed by 52 samples during 4-6 days of fever, and 47 samples during 7-9 days of fever.
Out of 56 samples (1-3 days of fever), 18 were positive by rapid NS1, 17 were positive by NS1 early ELISA, and 21 were positive by PCR. Out of 52 samples (4-6 days of fever), 15 were positive by rapid NS1, 15 were positive by NS1 early ELISA, and 22 were positive by PCR. Out of 47 samples (7-9 days of fever), 16 samples were negative while 31 samples were positive, of which 5 were positive by rapid NS1, 15 were positive by NS1 early ELISA, and 11 were positive by PCR [Table 1].
|Table 1: Percentage of positive results by rapid, ELISA, and PCR methods with duration of fever (n=155) |
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Comparison of each method with days of fever
Of 155 acute phase serum samples, positivity of rapid NS1, NS1 early ELISA, and PCR was 24.5% (38/155), 30.3% (47/155), and 34.8% (54/155), respectively [Figure 2].
|Figure 2: Test results of acute sera samples tested by Rapid, ELISA, and PCR test|
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The positivity of PCR (n = 54) slightly increased from 38.8% (21/54) on days 1-3 to 40.7% (22/54) on days 4-6 and decreased to 20.3% (11/54) on days 7-9. The positivity of rapid NS1 antigen (n = 38) decreased from 47.3% (18/38) on days 1-3 to 39.4% (15/38) on days 4-6 and further decreased to 13.1% (5/38) on days 7-9. Similarly, the positivity of dengue NS1 early ELISA (n = 47) also decreased from 36.1% (17/47) on days 1-3 to 31.9% (15/47) on days 4-6 but remained the same 31.9% (15/47) on days 7-9 [Figure 3].
Sensitivity and specificity of the tests
The rapid NS1 antigen was compared with RT-PCR, with sensitivity of 55.5% and specificity of 92%. Furthermore, positive and negative predictive values for rapid NS1 antigen were calculated and shown to be 78.9% and 79.4%, respectively, with an agreement of 79.3%.
The NS1 early ELISA test was compared with RT-PCR, with sensitivity of 66.6% and specificity of 89.1%. Furthermore, positive and negative predictive values for the NS1 early ELISA test were calculated and shown to be 76.5% and 88.3%, respectively, with an agreement of 81.2%.
| Discussion|| |
Acute febrile illness comprises infection due to malaria, influenza, leptospirosis, scrub typhus, typhoid fever, and dengue, of which some infections necessitate specific treatment. As antibiotics will be required to treat bacterial diseases, knowing the viral etiology will help in avoiding unnecessary administration of antibiotics.
In view of high mortality and morbidity associated with dengue especially in tropical countries, ,, it is imperative to diagnose the disease during the early phase in order to provide information for appropriate management and avoidance of complications. The NS1 antigen is found together with endothelium, free or soluble in the serum of patients, and can be detected on days 0-9 after the onset of symptoms. ,
A study by Dussart et al. showed that the NS1 antigen assay was more sensitive with samples collected up to day 3 after the onset of symptoms.  Similarly, in this study, rapid NS1 antigen and NS1 early ELISA techniques showed the highest percentage of detectable NS1 antigen of 47.3% and 36.1%, respectively, on days 1-3 of the acute phase sera.
On days 4-6, the positivity of rapid NS1 antigen was 39.4%, which reduced to 13.1% on days 7-9. Meanwhile, the positivity of NS1 early ELISA remained the same 31.9% on days 4-6 and days 7-9. The study by Kumarasamy et al. stated that the dengue early ELISA has the added advantage of giving good detection rates up to 7 days of the illness. 
In this study, the RT-PCR amplification yielded the highest percentage of positive results of 40.7% on days 4-6, which was raised from 38.8% on days 1-3 but reduced to 20.3% on days 7-9. In contrast, a study by Pok et al. demonstrated 100% sensitivity and specificity by RT-PCR assay within the first 3 days of fever; however, the positivity declined over time with 45.8% on days 5-6 and 8.3% on days 7-8 in the serum collected in the acute phase. 
Sensitivity and specificity tests are essential for accurate laboratory diagnosis of DENV-infected patients. Sensitivity of the NS1 ELISA test amounted to 66.6% and sensitivity of the rapid NS1 antigen amounted to 55.5%; of these, the ELISA technique appears to be a more sensitive method. A study by Najioullah et al. showed that the sensitivities of NS1 ELISA and rapid NS1 antigen were 61.2% and 49.4%, respectively, when compared to RT-PCR.  Similar results were also seen by Shrivastava et al. Their evaluation showed an overall higher sensitivity rate of the Panbio dengue early ELISA kit over SD bioline dengue NS1 antigen rapid test for laboratory diagnosis of acute dengue infection.  In contrast, with rapid ICT test, though the ELISA assay takes longer, and requires a better-equipped laboratory and technical expertise, it is considered as the confirmatory test. 
The dengue NS1 antigen was not found in patients with Japanese encephalitis virus or yellow fever virus infections  thereby implying that there is no cross-reaction of dengue NS1 protein with those of other related flaviviruses. Thus, detection of NS1 has been a promising test to diagnose dengue in its early febrile stage. ,
The availability of commercial dengue NS1 antigen test kits has provided an additional laboratory diagnostic tool for early detection of DENV. Such tests may be used in laboratories that have limited resources, lack viral culture, or RT-PCR facilities.
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[Figure 1], [Figure 2], [Figure 3]
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