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BRIEF COMMUNICATION  
Year : 2014  |  Volume : 57  |  Issue : 1  |  Page : 81-84
Early dengue diagnosis by nonstructural protein 1 antigen detection: Rapid immunochromotography versus two the enzyme-linked immunosorbent assay kits


Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Puducherry, India

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Date of Web Publication17-Apr-2014
 

   Abstract 

Dengue is known for its serious life-threatening complications. New rapid kits available recently in India target circulating non-structural protein (NS1) antigen from day one onwards. The sensitivity and specificity of a newly introduced rapid combo kit against two conventional ELISA kits is assessed. The performance of this kit is quite satisfactory since excellent agreement of 94.26% was observed with particular reference to NS1 antigen detection among all three kits namely Rapid SD Bioline dengue Duo (SD Korea), InBios DENV Detect NS1 ELISA, USA and dengue Early ELISA, Panbio, Australia. The false positivity of the rapid kit is very low since its specificity as for as NS1 antigen detection is concerned is 98.33%. The use of combination kit helps to detect additional cases of dengue, which are negative for NS1 antigen but positive for IgM and/or IgG antibodies, thus facilitating early diagnosis in remote areas and small laboratorie

Keywords: Dengue combo rapid kit, dengue non-structural protein antigen, dengue serodiagnosis

How to cite this article:
Stephen S, Charles MP, Anitharaj V, Deepa C, Umadevi S. Early dengue diagnosis by nonstructural protein 1 antigen detection: Rapid immunochromotography versus two the enzyme-linked immunosorbent assay kits. Indian J Pathol Microbiol 2014;57:81-4

How to cite this URL:
Stephen S, Charles MP, Anitharaj V, Deepa C, Umadevi S. Early dengue diagnosis by nonstructural protein 1 antigen detection: Rapid immunochromotography versus two the enzyme-linked immunosorbent assay kits. Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Jan 22];57:81-4. Available from: http://www.ijpmonline.org/text.asp?2014/57/1/81/130905



   Introduction Top


Dengue is a life-threatening illness common in tropics and sub-tropics. Dengue is a notifiable disease in India, but its exact prevalence is difficult to quantify due to the frequency of epidemics which appear throughout the country. Although dengue serotype 2 is the most prevalent serotype over the past 50 years, serotypes 3 and 4 have appeared in some epidemics. [1],[2],[3],[4],[5] In Southern India, an alarming increase is noted during this year's dengue season. The rapid kits available till recently could detect only IgM and IgG antibodies. [6],[7],[8] The recently introduced dengue rapid kits rapid immunochromatography test (RICT) in Indian market are combination packs, which detect circulating non-structural protein (NS1) antigen, a highly conserved viral protein and IgM and IgG. [9] This study aims at analyzing the sensitivity and specificity of RICT kit for NS1 antigen detection so as to explore its suitability for regular use in any modest laboratory or primary health center.


   Materials and Methods Top


A prospective study was conducted from January to November 2012, in a Tertiary care Super Specialty Teaching Hospital. Informed consent was obtained from patients before collecting blood for all laboratory investigations. A total of 554 blood samples were sent for dengue rapid test. Out of these, 140 clinically suspected cases of dengue which were also seropositive by RICT were taken up. Our physicians follow WHO criteria for clinical diagnosis of dengue. [10] Brief demographic details of our patients are added in the form of a separate table [Table 1].
Table 1: Demographic details of 140 dengue patients

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Inclusion criteria

Patients suspected for viral fever with temperature more than 37.5C with or without rashes and a positive tourniquet test followed by lab investigations such as low platelet counts.

Exclusion criteria

Those febrile patients with normal platelet count/hematocrit level or those who were positive for malaria after peripheral smear examination were excluded from the study.

Treatment of Dengue patients included bed rest, rehydration and other supportive measures. Platelet concentrates were administered only for those patients with very low platelet counts and seriously ill with hemorrhagic complications.

We included 40 controls - 20 from other PUO cases and 20 healthy blood donors.

The tests were performed strictly adhering to the kits' specifications.

RICT (SD BIO LINE dengue Duo-Standard Diagnostics, Korea) (dengue NS1+ Ab Combo)

The serum samples were first screened for NS1 antigen, IgM and IgG by SD BIO LINE dengue Duo (Standard Diagnostics, Korea).This rapid kit has two separate cassettes one for NS1 antigen and another for IgM and IgG antibody detection. The kits were observed for 20 min for appearance of control band along with the test band for NS1 antigen in the first cassette and IgM and IgG antibodies in the second. Sera were stored at –20°C for further testing by conventional ELISA for NS1 antigen by two commercial kits:

Dengue early ELISA Panbio, Australia

This is a single step antigen capture ELISA. NS1 antibody is coated in the wells. Once the NS1 antigen in patient's serum is bound, conjugated monoclonal antibody to NS1 antigen is detected by adding the tetramethylbenzidine (TMB) substrate. The reading was taken at 450 nm wavelength with 620 nm reference filter. The cut-off value, index value and Panbio units were calculated for each sample. Panbio units of ≥11 was taken as positive and ≤9 as negative. Panbio units of 9-11 were considered equivocal and were repeated twice.

InBios DENV Detect NS1 ELISA, USA

This is a two-step sandwich immunoassay. NS1 antibodies are bound to the wells. Patients' sera containing NS1 antigen are added along with a dilution buffer containing secondary antibodies. Once NS1 antigens are captured, colorimetric response is obtained by enzyme conjugate horseradish peroxidase and TMB substrate. The results were read at 450 nm. Cut-off values and immune status ratios (ISR) were calculated for the samples as per the instructions of kit. Samples with an OD of more than cut-off value were taken as reactive and less than cut-off were taken as non-reactive. Negative samples had ISR values ≤1 and positive samples ≥1. Borderline samples with ISR 0.9-1.1 were repeated in duplicate.

The inter/intra assay % coefficients of variation for RICT, InBios ELISA and Panbio ELISA were 3.0%, 3.4% and 5.4% respectively.


   Results Top


SD dengue Duo RICT kit detected 140 cases, out of which one child aged 10 years died. RICT results were classified into seven different groups depending upon the negativity/positivity of NS1 antigen either alone or with IgM and/or IgG in various combinations in RICT and compared with two NS1 dengue ELISA kits as shown in [Table 2].
Table 2: Comparison of seropositivity among 3 dengue kits (n = 180)

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Out of these 140 cases, 116 patients (82.86%) were positive by both RICT and InBios ELISA for NS1 antigen and 115 were positive by Panbio ELISA kit (82.14%). The lone sample missed by Panbio kit had both NS1 antigen and IgM antibody in RICT. All 40 controls were uniformly negative for NS1 antigen by the three tests. The cases peaked in the months of July to October and only few in March.

As could be inferred from [Table 1], the highest percentage of dengue cases belong to the age group of 21-40 years (48.6%), the lowest to the elderly 60-80 (1.4%) with the remaining two groups of 10-20 and 41-60 scoring equal 25% each.

Keeping InBios with the relatively higher sensitivity as the standard, the P values for RICT and Panbio were <0.5, which is statistically insignificant. Thus, the performance of all these three kits were equal.


   Discussion Top


There are four antigenically distinct serotypes of dengue virus: 1, 2, 3 and 4. The immunity being serotype specific, patients once infected with one serotype could at any time be exposed to other three serotypes. Such secondary infections can lead to dengue hemorrhagic fever and dengue shock syndrome with serious complications. The global prevalence of dengue has increased, affecting many countries. The aim of this preliminary study is to assess RICT kit's sensitivity and specificity in comparison to conventional ELISA as far as NS1 antigen detection is concerned. Dengue infection is serologically diagnosed by IgM ELISA in acute primary infection and IgG ELISA in late primary and secondary infections. In acute secondary dengue, IgM levels are significantly low, which might give rise to false negative results. Conventional ELISA is time consuming, need testing in batches and not suitable for small laboratories. The first reason for the lesser sensitivity and/or specificity of IgM and IgG detection could be a cross reaction among the antibodies of dengue virus with other flavivirus group. The other reasons are that it cannot differentiate between the past and present infection. Among the serological markers, NS1 antigen appears to be a better and earlier marker appearing between 1 and 9 days. The NS1 antigen is also highly sensitive and specific which does not cross react with Japanese B encephalitis and yellow fever. [11] However, it is emphasized that NS1 antigen has low sensitivity in case of secondary infection. The reason for this can be immune complex formation by NS1 antigen with the pre-existing antibodies. [12] The combination of both antigen and antibody detection by the same kit, solves these problems and aids in a better diagnosis. In our study all our RICT positive sera were positive in Panbio and InBios ELISA. Recently Arya et al.[2] detected 678 cases positive by NS1 antigen employing the same SD duo rapid kit. They reported that out of 678 dengue patients, 394 were positive for NS1 antigen alone and the remaining 284 were positive for NS 1 antigen plus IgM and/or IgG.

Our study shows 94.26% concordance among all the three kits in detecting NS1 antigen. Comparing with In Bios and Panbio ELISA, the sensitivity of RICT was 95.31% 97.54% respectively. Regarding specificity, while InBios ELISA and SD RICT are identical (100%), with reference to Panbio ELISA, SD RICT has 98.33% specificity. Eleven sera had only IgG antibodies in RICT and probably represent late primary or secondary dengue cases, which were negative for NS1 antigen in all three kits. Among 24 samples which were negative for NS1 antigen by SD RICT, 6 were positive by InBios ELISA. Incidentally, five of them had IgM and IgG antibody and one had IgM antibody in RICT. Panbio ELISA kit could pick up only four additional NS1 antigen positive cases (i.e., two less than InBios) out of which three had IgM and IgG and one had IgM in RICT. The reason for non-detection of NS1 antigen could be due to the immune complex formation. Attempt to dissociate the complex can solve this problem. [12] Kits targeting NS1 antigen may still give false negative report, if the quantity is less. A similar study comparing SD Bioline Rapid test, with other two ELISA (Platelia dengue NS1 antigen test and Pan E dengue Early ELISA) showed that rapid test had sensitivity of 80.7% and the ELISA-format tests had comparable sensitivities between 68% and 71% and specificity between 92% and 94% respectively. [13] According to Hang et al. the sensitivity of Platelia NS1 ELISA and NS1 rapid lateral flow rapid test were 82% and 72% respectively, whereas both tests had a specificity of 100%. [14] A total agreement of 90.1% between the NS1 antigen lateral flow kit (SD Bioline) and Panbio NS1 antigen capture ELISA was observed by Shrivastava et al., according to whom, compared with Panbio dengue ELISA the sensitivity of SD NS1 antigen by lateral flow technique was only 62.5% but the specificity was 100% [12] Dussart et al. tested 222 acute sera for NS1 antigen with a rapid kit, (Biorad France), Platelia dengue NS1 antigen ELISA (Biorad) and Pan E dengue early ELISA Panbio, Australia. The result was a sensitivity of 81.5%, 87.4%, 60.4% and specificity of 100%, 100% and 97.9% respectively. [15] Tontulawat et al. considers rapid dengue NS1 antigen along with IgM antibody as highly appropriate for diagnosis of dengue. [16] Watthanaworawit et al. tested 162 samples and reported that the sensitivity of real time reverse transcriptase polymerase chain reaction (PCR), NS1 antigen ELISA and IgM antibody ELISA were 89%, 54%, 17% and specificity was 96%, 100% and 88% respectively. [17] Thus most of the workers reported almost similar specificity between Rapid dengue kit for NS1 antigen and other conventional ELISA kits. However, the sensitivity of the rapid kits were low to moderate. In our observation, the SD RICT is matching two conventional ELISA tests, both in sensitivity and specificity with 95.4% and 98.33% respectively. Thus, the false positivity shown in SD RICT is very minimal (1.67% only).

To help the physicians and patients, only the rapid test RICT was carried out as a screening test. The cases peaked in the months of July to October and only few in March. All the 140 positive sera were kept frozen at -20°C. InBios ELISA and Panbio ELISA for these 140 patients were performed within 20 days during 16 th November-4 th December, 2012. Forty negative controls were tested by all three kits during these 20 days.

Regarding the affordability, the rapid kit which screens for three parameters namely, NS1Ag, IgM and IgG costs Rs. 400/- per test. The InBios ELISA and Panbio ELISA with 96 well formats with a single parameter of NS1 Ag alone costs Rs. 150/- per test.

Advantages and disadvantages of Rapid RICT kit over conventional ELISA kits

Compared with conventional ELISA which needs 4 h, RICT results are available within 20 min. This will be very helpful in initiating immediate treatment and minimizing the serious complications and mortality of dengue. Conventional ELISA cannot be performed for singe or small number of samples, since it would be quite uneconomical. Hence, they are used for batch testing of large samples and single plate can be used once with 92 samples or twice with 44 samples each time.

The limitations of Rapid kit are their relatively lower sensitivity and specificity (90%). They are quite susceptible to unfavorable storage conditions and can give false results.

If not intervened at an early stage, dengue can cause serious complications. RICT which is an easy test can be used in primary care centers so that early diagnosis can be made and morbidity and mortality could be significantly brought down. Performing three conventional ELISA for NS1 antigen, IgM and IgG, for each patient would face lot of practical difficulties. Since dengue is a notifiable disease, introduction of RICT combination packs in all levels of health care system for speedy and accurate diagnosis and confirmation of the clinical suspicion would be the need of the hour. This would also ensure initiation of control measures by the public health authorities. However there are two riders to this recommendation:

  1. Cross reactions are possible with other diseases such as scrub typhus, tuberculosis, leptospirosis, malaria etc. Many samples from such diseases should be included and a meta-analysis is needed.
  2. Periodic quality control checks of these rapid kits and maintaining appropriate storage conditions are two very essential criteria to be strictly adhered to, so as to avoid false positive or false negative results. Storage conditions are critical especially in remote/rural areas. Strict adherence to instructions outlined in the technical brochures is essential. Whenever any aberrant results are obtained invalidating the assay, a comprehensive review of standard operating procedures followed by the laboratory personnel is to be initiated so as to look for any procedural deviations, which caused the error.


In the early febrile phase, detection of NS1 antigen is an important test as confirmed by several workers. [1],[2],[3],[4],[18],[19],[20],[21] However, interpreting results clinical correlation is essential and where facilities exist, additional tests like PCR and virus isolation may be recommended.


   Acknowledgments Top


The authors express their sincere thanks to the Chancellor, Vice Chancellor and Dean of Mahatma Gandhi Medical College and Research Institute, Pondicherry for the encouragement and support provided for this research work.

 
   References Top

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3.Datta S, Wattal C. Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection. Indian J Med Microbiol 2010;28:107-10.  Back to cited text no. 3
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4.Singh MP, Majumdar M, Singh G, Goyal K, Preet K, Sarwal A, et al. NS1 antigen as an early diagnostic marker in dengue: Report from India. Diagn Microbiol Infect Dis 2010;68:50-4.  Back to cited text no. 4
    
5.Padbidri VS, Wairagkar NS, Joshi GD, Umarani UB, Risbud AR, Gaikwad DL, et al. A serological survey of arboviral diseases among the human population of the Andaman and Nicobar Islands, India. Southeast Asian J Trop Med Public Health 2002;33:794-800.  Back to cited text no. 5
    
6.Blacksell SD, Mammen MP Jr, Thongpaseuth S, Gibbons RV, Jarman RG, Jenjaroen K, et al. Evaluation of the Panbio dengue virus nonstructural 1 antigen detection and immunoglobulin M antibody enzyme-linked immunosorbent assays for the diagnosis of acute dengue infections in Laos. Diagn Microbiol Infect Dis 2008;60:43-9.  Back to cited text no. 6
    
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10.WHO. Dengue: Guidelines for Diagnosis, Treatment, Prevention, and Control in sub-Saharan Africa and 13 Countries in South America. Geneva: World Health Organization; 2009.  Back to cited text no. 10
    
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14.Hang VT, Nguyet NM, Trung DT, Tricou V, Yoksan S, Dung NM, et al. Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis 2009;3:e360.  Back to cited text no. 14
    
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16.Tontulawat P, Pongsiri P, Thongmee C, Theamboonlers A, Kamolvarin N, Poovorawan Y. Evaluation of rapid immunochromatographic NS1 test, anti-dengue IgM test, semi-nested PCR and IgM ELISA for detection of dengue virus. Southeast Asian J Trop Med Public Health 2011;42:570-8.  Back to cited text no. 16
    
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Correspondence Address:
Selvaraj Stephen
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondy-Cuddalore Main Road, Pondicherry 607402
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.130905

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    Tables

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