Diagnostic Efficacy of Rapid Immunochromatographic Test in Diagnosis of Dengue Infection

Dengue is a mosquito-borne arboviral disease of grave public health concern worldwide. Early diagnosis and treatment is required to reduce morbidity & mortality from complications caused by secondary dengue infection. According to WHO, the three main diagnostic modalities for the diagnosis of dengue infection are cultivation and identification of viruses, molecular methods, and serology. Whereas virus cultivation is labour intensive and available only in reference laboratories, molecular methods require expensive infrastructure & expertise. Serology on the other hand not only less tedious but is also able to differentiate between primary and secondary dengue. This study was undertaken to evaluate the diagnostic efficacy of rapid immunochromatographic assay in the diagnosis of dengue infection as compared to ELISA. The study was conducted in the serology section of the Microbiology laboratory, Sri Guru Ram Das Institute of Medical Sciences, Amritsar. Blood samples from 429 patients with clinical suspicion of dengue virus infection were received in the lab from August 2020 to December 2020. All samples were subjected to rapid ICT and ELISA to detect NS1 Ag and IgM antibodies. The majority number of cases were observed in the age group of 31 to 40 years while the gender-wise ratio was 1.43:1 showing male preponderance. Out of 429 samples tested, 156 were reactive for either NS1 antigen or IgM antibodies by the ELISA method. Results of rapid ICT for NS1Ag and results of NS1Ag by ELISA were analyzed and compared. A sensitivity of 81.25% was noted and specificity of 100%. IgM detection by rapid ICT in comparison to IgM ELISA shows a sensitivity of 82.14% and specificity of 100%. Rapid ICT kits performed at par with the ELISA. Rapid immunochromatographic assays are important diagnostic tools in the identification of dengue and early treatment of dengue patients is possible, reducing mortality significantly.


iNtRODUCtiON
Dengue also called break-bone fever, is caused by a virus belonging to the genus Flavivirus which is an Arbovirus. 1 It is a mosquito-borne virus.Female mosquitoes mainly Aedes aegypti and Aedes albopictus are the vectors of this virus. 2hese mosquitoes are vectors for some other diseases like zika fever, yellow fever, chikungunya etc. 1 Dengue virus is from family Flaviviridae.It has four serotypes namely DENV-1, DENV-2, DENV-3, and DENV-4, which are responsible for causing Dengue.Infection of one serotype provides lifelong immunity against that specific serotype but there is no or very less cross-immunity to other serotypes. 3econdary infection by other serotypes leads to the development of serious disease e.g dengue hemorrhagic fever. 1 Pre-existing dengue antibodies are liable for severe disease.Within few days of the second dengue infection, Virus-antibody complexes are formed.A large number of mononuclear cells are infected by these non-neutralizing, enhancing antibodies, which result in the release of cytokines, vasoactive mediators, and procoagulants causing disseminated intravascular coagulation which causes dengue hemorrhagic fever syndrome. 4round 3.9 billion of the population are under threat of dengue infection worldwide, maximum risk in the tropical region especially Southeast Asia and western pacific. 1 An early diagnosis of the Dengue virus should be done for rapid treatment of the disease and also rule out other differential diagnoses.According to WHO virological and/or serological tests must be performed for the diagnosis of dengue.Virological methods include isolation of the virus, virus nucleic acid detection by RT-PCR.Whereas serological methods include identification of a protein, NS1 antigen which is produced by the virus.Other than the ELISA method, which according to WHO is the diagnostic test for diagnosis of dengue, Rapid diagnostic kits, which are immunochromatographic tests (ICT) assay are also available for this.These rapid ICT assay do not require trained staff or costly and highly equipped labs.These rapid ICT assay give results within 20 minutes.Serological methods also detect IgG and IgM anti-dengue antibodies.IgM antibodies are detectable 1 week after infection and are highest at 2 to 4 weeks after the onset of illness suggesting a recent infection.][7] Hence either the rapid ICT assay or ELISA test can be performed to detect the presence of NS1 or Ig M antibodies in the serum of the patient.Rapid diagnostic tests which are non-ELISA-based immunochromatographic tests (ICT) are less reliabale. 8There are different reports regarding sensitivity and specificity of ICT.This study aims to determine and evaluate rapid diagnostic test for NS1 Ag and IgM antibody for diagnosis of dengue in comparison to ELISA tests in terms of diagnostic efficacy, sensitivity, and specificity of rapid ICT kits used in our hospital.

MATERIALS AND METHODS
The present study was carried out at the Microbiology department, Sri Guru Ram Das Institute of Medical Sciences, Amritsar, during the 2020 dengue season (August; 2020 to December; 2020) in Amritsar, India.Blood samples of 429 patients suspected of DENV infection were received in the lab.These patients were of all age groups.Serum was separated from these samples and further analyzed.Each serum sample was tested for both rapid immunochromatographic assay (SD Biosensor Dengue Ag +Ab Duo) manufactured by SD Biosensor Healthcare Pvt. Ltd., India which detects NS1 antigen, IgM and IgG antibodies and ELISA (ERBA Lisa Scan EM & Lisa Wash) for detecting NS1antigen and IgM Capture Elisa for IgM antibodies.Data regarding NS1 antigen and IgM antibodies were compiled and analyzed statistically.

Ethical Clearance
Approval from the ethical committee of the institute was obtained for the study.Ref No.SGRD/IEC/2021-07

Statistical Analysis
Data collected was analyzed with MedCalc statistical software version 20.0.4.

ResUlts
A total of 429 samples of suspected dengue patients were received in the microbiology lab.The age group that showed the highest seropositivity was 31 to 40 years, next to this group was 21 to 30 years age group.(Table 1).Genderwise distribution is shown in Table 2. Out of total samples tested male to female ratio was 1.43:1 showing male preponderance.
Out of 429 samples tested, 156 samples were reactive for either NS1 Ag or IgM antibodies by the ELISA method and 127 were found to be positive for dengue infection by rapid ICT assay.The samples tested with rapid ICT and IgM ELISA and comparison of these tests showed that of the total 429 samples, 127 were positive and 301 were negative by both the tests.There were 24 samples which were positive by ELISA and negative by rapid ICT for NS1 Ag and 5 samples were positive by ELISA and negative by ICT for IgM and were considered false negative.
The sensitivity of the rapid ICT assay for NS1Ag was 81.25% and it was 100% specific.The positive predictive value of rapid ICT assay for NS1Ag is100% and a negative predictive value of 92.61% was noted for the same, while data accuracy was calculated to be 94.40%.(Table 3) Table 4 depicts a comparison of IgM detection by rapid ICT with IgM ELISA, showing sensitivity 82.14%, specificity 100%, positive predictive value 100%, and negative predictive value of 98.76% while data accuracy of 98.83% was observed.

DisCUssiON
Dengue is prevalent worldwide, but southeast Asia and Latin America are the hot spots of dengue.Other febrile illnesses caused by mosquito-borne viruses like Zika or Chikungunya have similar signs and symptoms which resemble those of dengue.Some of such symptoms are fever, myalgias, maculopapular rash, retro-orbital pain, and lymphadenopathy.Thus making it difficult to diagnose without laboratory investigations. 5Many rapid immunochromatographic test assays for detecting dengue NS1, IgM and IgG antibodies are available and many studies have evaluated their performances.In this study, the performance of ICT (SD Biosensor Dengue Ag +Ab Duo) used in our hospital in comparison to MAC-ELISA (ERBA Lisa Scan EM & Lisa Wash, Germany) was compared.
In this study 429 serum samples of suspected dengue patients were tested by rapid ICT and ELISA for both NS1 antigen and IgM antibodies.The foremost common cohort affected was 20 to 40 years.Similar results were given by Sarah Hassan et al. 9 Also these results are in line with the study by Garg et al. 10 which observed that the median age was 35.5 years.T h e s t u d y a l s o s h o w e d m a l e predominance, Garg et al., 10 Tabasum MB et al., 7 Annapurna SL et al., 11 observed similar results while on the contrary Anand AM et al., 3 showed male to female ratio of 1:1.35.
In our study, rapid ICT assay for NS1 Ag and IgM shows sensitivity more than 80% and specificity 100% compared to ELISA.Another study by Reddy RM showed over 90% sensitivity and over 98% specificity 12 While studies conducted in various hospitals recorded a good range of sensitivity of rapid ICT for NS1 Ag 49.4%-98.9%and specificity starting from 91%-100%. 13ensitivity of 82.14% and specificity of 100% were observed in our study for IgM antibody detection by rapid ICT in comparison to ELISA during this study.These results are slightly different from those observed in another study by Hassan S et al. 10 While a study conducted in Srilanka shows a sensitivity of 64% and specificity of 74.7% of anti-DEN V IgM detection by rapid ICT. 14 This difference in reported sensitivity and specificity could be because of different commercial assays used in different studies. 11In the present study 100% Positive predictive value of rapid ICT for NS1 Ag and IgM is noted.Positive predictive value is the probability that subjects with a positive screening test truly have the disease so there was no need for confirmatory testing.Similar findings were noted by several other authors Garg et al., 10 Shih et al. 15 The study shows that the sensitivity of rapid card test is less but has a good specificity.In situations of epidemic, the card test can be used for screening but with the support of IgM capture ELISA. 16irus cultivation is labour intensive and available only in reference laboratories while molecular methods require expensive infrastructure & expertise.Serology on the other hand not only less tedious but is also able to differentiate between primary and secondary dengue.
Rapid Immunochromatographic test assay is an easy and simple procedure, no special equipment or no trained staff is required This test is cost-effective because many samples are to be put together for ELISA to be cost-effective, which increases its turnaround time.Rapid ICT assay can be performed on a single sample as soon as the sample is received within the lab.Rapid ICT results are available within 15 to 20 minutes.Our study found that rapid ICT assay that we used for NS1 Ag and IgM performed at par with ELISA-based tests.But in step with the definition given by WHO of confirmed cases, all cases which are screened by rapid ICT assay should be confirmed by ELISA. 1

CONClUsiON
In resource-limited countries, which lack diagnostic laboratories with types of equipment for PCR, virus isolation, or ELISA, esp in remote and rural areas, rapid ICT assay is an important diagnostic test of dengue and early management of such patients is feasible, reducing the mortality significantly.

Table 2 .
Gender wise distribution of dengue positive case

Table 3 .
Comparison of ELISA and rapid ICT for NS1 Ag