Quantitative Estimation of Dengue NS-1 Antigen by Enzyme-Linked Immunosorbent Assay with its Clinical and Laboratory Correlation – An Observational Study

INTRODUCTION

Dengue fever, a flavivirus infection spread by the Aedes mosquito, is the most prevalent and widely spread viral haemorrhagic fever in the world, mainly in the Americas, Pacific islands and continental Asia.¹ There are four serotypes of dengue virus that is, dengue (DEN) 1 to DEN 4. Three structural proteins are encoded by genomic RNA: C (core protein), M (membrane protein) and E (extracellular protein) (envelope protein) while non-structural (NS) 1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 are the NS proteins. NS1 is a highly conserved glycoprotein that is required for virus viability but has yet to be recognised as having any biological action.² NS1 can be detected during the acute phase of dengue virus infections, and NS1 tests are recommended because they can be as sensitive as molecular tests within the first 0–7 days of symptoms. However, NS1 tests are not advised after day 7. A positive NS1 test result indicates dengue infection, but it does not provide serotype information, which is only required for surveillance. While NS1 can be found in whole blood or plasma, the majority of NS1 tests have been developed and evaluated on serum samples. Although combined testing with NS1 and immunoglobulin M (IgM) antibody tests can provide a diagnostic result within the first 1–7 days of illness, if both antigen and antibody tests are negative, a second convalescent-phase specimen should be obtained and tested for IgM.³

Dengue fever has been on the rise for decades, and the true number of cases is significantly underestimated. Globally, it is estimated that 390 million individuals are infected, with 96 million developing clinical illness,⁴ producing major morbidity and mortality, particularly in low- and middle-income countries and posing a significant economic burden. Asia is accountable for 70% of the disease burden. Dengue fever has surged by more than eightfold in the last two decades, affecting the younger population predominantly.^5,6

Dengue infections must be diagnosed rapidly and accurately to reduce patient morbidity and mortality. This study was conducted to aid in the quick diagnosis of this critical illness by quantitative approaches that were missed by qualitative methods for early diagnosis, disease surveillance and timely management to save lives.

Therefore, this study aimed to evaluate the quantitative estimation of dengue NS1 antigen, estimate the proportion of dengue at a tertiary care hospital, correlate the quantitative dengue NS1 with other laboratory parameters and clinical presentation, associate the quantitative levels of NS1 antigen with dengue complications and associate the quantitative levels of NS1 antigen with the mortality rates.

MATERIAL AND METHODS

An observational cross-sectional study was conducted for 18 months within a tertiary care centre, following approval from the Institutional Ethics Committee (IEC/DISS/17118). The study focussed on evaluating dengue symptoms among patients presenting at various outpatient departments and inpatient departments of the hospital. A sample of 280 symptomatic individuals underwent preliminary assessment through qualitative enzyme-linked immunosorbent assay (ELISA) before comprehensive evaluation. Blood samples were procured from suspected dengue cases subsequent to obtaining informed consent. Exclusion criteria encompassed individuals testing positive for leptospira IgM antibody, malaria or chikungunya IgM antibody. Patient-specific parameters such as age, gender, symptoms, comorbidities, total leucocyte count, platelet count and risk categorisation were recorded. A comprehensive clinical history, along with pertinent test data, was collected utilising a standardised case record form. Data collation was executed through Microsoft Excel, followed by subsequent statistical analysis including Pearson Chi-square, Shapiro–Wilk test, Spearman’s rho correlation and Z-statistics.

RESULTS

During the study period, 280 symptomatic individuals over the age group two to 72 years [Table 1] with 68% males and 32% females formed our study sample. Patients mostly presented with fever (100%) chills (95%.7) and body aches (95.4) [Table 1]. Hypertension was seen in 2.1% of the patients while most of them had no associated comorbidities (96.8%) [Table 1]. Bleeding tendencies (4.3%) were the most commonly observed clinical complications in patients [Table 1]. With 98% of the patients discharged, death was observed in only 2% of the patients. For the qualitative ELISA test, 86.8% of patients were found to be positive, 92.1% of patients were positive for quantitative ELISA whereas rapid antigen test showed 96.1% positive results. All the parametric variables (TLC, platelets count, pulse pressure [mm Hg], SGOT [IU/L], SGPT [IU/L]) failed the normality test with Shapiro–Wilk test and was found to be statistically significant for all these variables (P < 0.05). Spearman rho correlation was performed between the parametric variables and quantitative NS1. Negative and statistically significant (P = 0.001) correlation was observed between quantitative NS1 and the parameters including TLC, platelet count, pulse pressure and SGOT whereas a positive and statistically significant (P = 0.024) correlation was found between quantitative NS1 and SGPT [Table 2]. No statistically significant association was seen between the complications and quantitative ELISA test result (P = 0.110) [Table 3]. A statistically significant association was seen between the mortality rate and quantitative ELISA test result (P = 0.040) [Table 4], whereas no statistically significant association was seen between the clinical outcome and quantitative ELISA test result (P = 0.510), which showed that clinical outcome was independent of the quantitative ELISA test results [Table 5].

DISCUSSION

Dengue fever is one of the most serious infectious diseases, wreaking havoc on public health in many tropical and subtropical nations. The chance of acquiring a secondary dengue infection is directly proportional to increasing age. Aging also impairs physiological functions affecting the immune system and increasing the presence of chronic diseases.⁷ In a study by Gitika et al. where a little over half of the patients (52%) were in age group of 21–40 years, 14% of patients were <10 years, 24% of patients belonged to 41–60-year age group and >60-year age group showed 10% of patients. The mean age was 37.31 ± 17.54 years. Most of these patients were adults because they form the working-age group and are more exposed to insect bites⁸ which is in similarity with our study having most patients within the age group 21–40 years. Another similar study was done by Savargaonkar et al. in which the most afflicted age group was found to be between 11 and 30 years.⁹

In a study conducted by Muhammad et al., where dengue fever incidence in 95 patients was observed, they found 81.1% males and 18.9% females.¹⁰ This is in similarity with the gender differences in our study that is, male 68% and female 32%. In another similar study by Tahlan et al., a male-to-female ratio of 2:1 was observed. This might be due to a reason that males predominantly form the working population and more prone to infection by mosquito bites in the daytime.¹¹

Fever had been identified as the most prevalent presenting symptom by Neeraja et al. and Shah et al. in 100% of their study population. However, Ghosh et al. in his study found 71% of patients presenting with fever and chills.^12-14 Jelinek et al. state the symptoms commonly associated with dengue, such as fever (86%), myalgia (85.6%), headache (59.2%) arthralgia and exanthema can help make the diagnosis, when present, but the absence of typical symptoms does not exclude infection. Most patients with dengue in this study were symptomatic and reported fever.¹⁵

When compared to cases without comorbidities, comorbidities elevated the case fatality rates (CFRs) of hospitalised dengue by 3-–17-fold. Furthermore, independent of dengue severity or age, the CFRs for hospitalised dengue were greater in the presence of common comorbidities.¹⁶ In a study by Lee et al., the impacts of coexisting comorbidity(ies) and glycaemic control on dengue severity were emphasised. As there were no comorbidities associated among the subjects who had tested positive and negative for the ELISA test, the findings mentioned above can be related to our study.¹⁷

Godbole et al., in their study, found pleural effusion and ascites in 11% of the dengue patients. About 11% of the individuals experienced bleeding, which is greater than the findings from our study (4.3%).¹⁸ Numerous studies have pointed out common and uncommon complications associated with dengue fever. These include acute respiratory distress syndrome, encephalopathy and encephalitis, swollen lymph nodes, enlarged spleen, myocarditis, anaemia, multiple organ failure, hepatitis, fever, refractory shock, portal hypertension, pericardial effusion, muscle inflammation (myositis), acute kidney injury and disseminated intravascular coagulopathy.^19-21

Eight patients (11.1%) died in a study by Chandralekha et al. of 72 critically ill dengue patients.²² In their investigation, Juneja et al. reported a mortality rate of 6.1%.²³ In a multicentre study by Schmitz et al., 42 individuals in India who were brought to the intensive care unit (ICU) with dengue haemorrhagic fever or dengue shock syndrome reported 8 in-ICU deaths, resulting in a 19% mortality rate.²⁴ In-ICU and in-hospital death rates were reported to be 18.6% and 19.6%, respectively, in a study by Amâncio et al.²⁵ The variations in disease severity could be the cause of the differences between our study results and other findings.

Many studies have been conducted to investigate the utility of liver transaminase levels, platelet counts and other clinical and laboratory parameters in predicting severe dengue, and it has been demonstrated that none of these parameters can be used alone to predict severe dengue.^26-29 According to Paranavitane et al., NS1 antigen levels are significantly and inversely related to all white blood cell parameters. Serum NS1 antigen levels were found to be significantly (P = 0.0001) and inversely related to total white cell and lymphocyte counts.³⁰ However, according to Ju and Brasier, platelet and lymphocyte counts, as well as serum interleukin-10 levels, were the most important variables associated with severe dengue.³¹ Although Duyen et al. did not correlate the kinetics of NS1 antigen levels with overall clinical disease severity, they did show that higher NS1 antigen levels on day 3 of infection were associated with lower platelet counts.³² Similarly, a negative and statistically significant correlation was observed between quantitative NS1 with TLC, platelet count, pulse pressure and SGOT.

CONCLUSION

This study underscores the significance of early and precise diagnosis in managing dengue fever. Our findings highlight the utility of quantitative estimation of dengue NS-1 antigen by ELISA, demonstrating strong correlations with clinical and laboratory parameters. The high positivity rates observed with quantitative ELISA NS1 tests, alongside the notable mortality rates, emphasise the severity of dengue infections. Importantly, the significant correlations observed between NS1 antigen levels and various clinical indicators, such as total leucocyte count, platelet count and liver enzymes, provide valuable insights into disease progression and prognosis. These results underscore the importance of comprehensive patient evaluation and monitoring in mitigating the adverse outcomes associated with dengue fever. Further research and clinical validation are warranted to enhance our understanding of dengue pathogenesis and refine diagnostic and management strategies to improve patient outcomes.