INTRODUCTION:

The Arbovirus, which belongs to the Flavivirus family, causes Dengue, a mosquito-borne disease that belongs to the Flavivirus genus. It is often referred to as break-bone fever as well. Female Aedes aegypti and Aedes albopictus mosquitoes are the predominant carriers of this virus¹. In accordance with the World Health Organisation (WHO), the dengue virus infects around fifty million individuals every year, of which roughly three billion reside in regions where the virus is prevalent globally. These viral infections represent about 500,000 hemorrhagic dengue fever cases, and the disease claims the lives of 25,000 individuals worldwide each year². The main signs and symptoms of a dengue virus infection include a fever and rash that cover the entire body, an increase in body temperature that lasts for two to seven days, excruciating pain in the tendons, muscles and joints and retro-orbital pain, headaches, nausea, and vomiting³. Skin haemorrhages, or bleeding beneath the skin's surface, can cause purple or red dots form on the entire body in dengue fever patients⁴. The fever is extremely high during this period, and the number of thrombocytes in the blood decreases over the course of the next few days⁵. Based on types of antigens, dengue is transmitted by four diverse serotypes of the virus: DENV-1, DENV-2, DENV-3, and DENV-4^6,7. Out of four known serotypes of dengue, serotype 4 (DENV-4) was the first to be discovered in Brazil.

A precise diagnosis is crucial for selecting the correct method of treatment. A high body temperature persisting for at least seven days is required for a clinical diagnosis in accordance with World Health Organisation (WHO) guidelines⁸. The methods that are employed for confirmatory dengue laboratory diagnosis include the detection of ribonucleic acid (RNA) of dengue virus isolation in culture, serological assays for NS1, IgM, or IgG seroconversion, and antigen detection^9,10. These diagnostic techniques are costly, time-consuming, and require specialised staff and apparatus. These factors make them potentially impracticable, particularly in times of epidemics. IgM/IgG antibody detection can be done with commercially available immunochromatographic and ELISA techniques, which can provide results in the span of minutes to hours^11,12. However, an individual affected by dengue cannot recognise antibodies until 4-5 days after the disease onset. The dengue virus infection is characterised by an increase in the level of a highly conserved glycoprotein identified as dengue NS1 antigen, which produce in both secretory form and also attached with membrane in the early stages of dengue infection in all serotypes^13,14. The NS1 antigen can be detected before the formation of antibodies^15,16. From the first day following the start of the fever until Day 9, the dengue specific antigen NS1 can be detected in the blood^17,18. Conversely, three to five days following the onset of symptoms, an increase in particular immunoglobulin M (IgM) levels is indicative of a primary dengue virus infection. IgG levels are detectable for the entirety of an individual's life and begin to rise even after 10 to 14 days¹⁹.

In order to treat the disease quickly, an early diagnosis of the dengue virus is necessary, as is ruling out other possible causes. Rapid immunochromatographic tests (RITs) are an appropriate choice for early detection in low-resource settings, can be utilized at the point of treatment, and do not require specialised infrastructure or technical skills²⁰. RITs are currently being used more frequently in developing nations for identifying infectious epidemics, where they can be carried by medical professionals in clinics or by untrained health workers in communities²¹. This study aims to determine and evaluate the diagnostic effectiveness, sensitivity, and specificity of the RITs for dengue fever in relation to ELISA tests combining NS1 Ag and IgM/IgG antibody rapid diagnostic tests.

MATERIALS AND METHODS:

Population study and collection of sample:

The present examination was conducted from April 2023 to November 2023 in the Department of Medical Laboratory Technology at the Shri Alpesh N. Patel PG Institute of Science and Research, Anand (Gujarat) and collaborated with the Department of Pathology, Rytham Hospital, Khambhat, Anand (Gujarat) to undertake a comparative study. The Institutional Ethical Committee at the Shri A. N. Patel PG Institute of Science and Research, Anand (India) approved the study protocol by the Declaration of Helsinki (Approval Date: March 2, 2023; number: P-02). No private or confidential information was collected. Local Institutional Ethics Committee. Documented consent was received from all patients following study explanation. A total of 155 acute-phase blood samples were collected from children, adults and aged ranging in age from 0 to 60 years. During the research period, all patients (adults, children, and elderly) with fever who fit the new World Health Organisation (WHO) definition of dengue were included. Formal consent was obtained from the parents or caretakers of patients. In the laboratory, each patient was given two blood samples along with their full medical history, which included their IP number, name, and surname. For instance, one tube should be used for both the peripheral smear for the platelet count and the total blood count/picture. A haematology analyser (Mindray BC 5140 5 Part) was used every day to perform whole blood counts or platelet counts²².

Immunochromatographic card assay.

After serum had been separated from all of the specimens, kits were utilised to test the presence of antibodies IgM/IgG and dengue specific antigen NS1. The objective of the Dengue fever rapid IgG/IgM immunochromatographic test is to qualitatively identify and separate specific IgM and IgG antibodies to the virus load present in human serum. This test is effective for distinguishing both primary and secondary dengue infections by detecting anti-dengue IgM and IgG antibodies. Tests that show only the control line (C) are regarded as negative. A result showing only the control line (C) is considered negative. A primary dengue virus infection is indicated if two lines (IgM and control) appear in the result window. A positive diagnosis for a secondary dengue virus infection is indicated if three lines—IgM, IgG, and control—are displayed in the result window. Dengue NS1 Ag Strip (The Bioline™ Dengue Duo kit) was used for the detection of NS1 antigen from serum. The reaction is carried out on an immunochromatographic strip by applying serum or plasma with a migration buffer²³.

NS1 Antigen Detection ELISA Assays:

The dengue NS1 Ag ELISA is the method used to detect the presence of NS1 antigen found on dengue virus in serum. This test is called solid-phase enzyme-linked immunosorbent assay to detect dengue NS1 antigen²⁴. The assay was performed strictly following the instructions provided by Abbott Bioline Dengue NS1 Ag Test Kit manual. Microwells with strong reactivity for the Dengue NS1 antigen are coated with anti-Dengue NS1 antibodies. The wells are filled with the serum sample. Then, an enzyme conjugate (monoclonal anti-dengue NS1 antibodies connected to HRPO) was introduced. Wash buffer is introduced to remove the unbound conjugate. The quantity of bound peroxidase is directly correlated with the sample's concentration of dengue NS1 antigen. A yellow colour appears when a stop solution is given to limit the enzyme-substrate reaction, and this colour is ultimately measured spectrophotometrically at 450nm.

Statistical analysis:

The statistical study was carried by utilising SPSS version 21 software. The result was considered statistically significant when P was less than 0.05.

RESULTS:

This study included 155 cases in total, of which 82 (52.9%) were male and 73(47.10%) were female. In this study, the patients' ages ranged from 3 to 56 years. The age-wise distribution has been tabulated in Table 1. The majority of dengue cases were reported in the 21-30 age group, with 68 cases. This was followed by the 31-40 age group, which had 38 cases. The fewest cases were observed in the 41-60 age group, with only 10 cases. Table 2 displays the frequency of clinical findings and total number of dengue-positive cases.

Table 1: Age and sex wise distribution of dengue positive patients

Age group (Years)	Male (n)	%	Female (n)	%	Total (n)
0-10	7	8.52	5	6.85	12
11-20	15	18.29	12	16.44	27
21-30	32	39.02	36	49.31	68
31-40	22	26.83	16	21.92	38
41-60	6	7.32	4	5.48	10
Total (n)	82		73		155

Table 2: Number of cases according to clinical sign and symptom in dengue positive cases

	Number of cases (n)	Percentage (%)
Fever	155	100
Headache	132	85.16
Myalgia	128	82.58
Rash	52	33.54
Pain Abdomen	75	48.39
Bleeding Manifestation	23	14.84
Vomiting	24	15.48
Diarrhea	14	9.03

IgM, IgG, and dengue NS1 antigen have been determined in all serum samples obtained for this investigation. Based on the presence of the IgM antibody and the NS1 antigen, 69.67% (108/155) of the 155 examined samples were determined to be positive for dengue virus infection. This was followed by the presence of the NS1 antigen (16.77%) (26/155), as shown in Table 3. Only one sample was found to have an IgG-positive antibody for dengue virus.

Table 3: Comparison of efficacy of various dengue specific parameters in the diagnosis of dengue infection using rapid test kit method on the day (Day 1) of admitted in hospital

Method of detection	Total number of cases (n)	Percentage (%)
NS1 only	26	16.77
NS1 + IgM	108	69.67
IgM only	12	7.75
IgM + IgG	8	5.16
IgG only	1	0.65

A total of 155 clinically suspected dengue cases (positive by one or more of the following tests: NS1, IgM, and IgG) were assessed further from day 1 to day 7 of fever. Figure 1 displays the sensitivities of IgM/IgG antibody and NS1 antigen detection in relation to the number of days following the beginning of fever. The sensitivity of NS1 antigen detection increased from day 1 to day 4 (145 cases showed positivity out of 155 on 4^th day). Afterwards, the sensitivity of NS1 declined up to the 7^th day. The sensitivity towards the positivity of the IgM antibody in all suspected dengue cases increased from day 1 to the 3^rd day and started to decline up to the 7^th day. The presence of IgG antibody positivity was very low in all cases.

Figure 1. NS1, IgM, and IgG positivity in all suspected dengue cases from the infection's onset date.

The sensitivity of the IgG antibody was drastically increased from the 5^th day of the onset of dengue fever. Five samples that tested positive by the NS1 rapidly immunochromatographic test method but negative by ELISA were considered to be false negatives. Ten samples tested positive by ELISA but negative by rapid RIT for NS1 Ag. Based on findings from the RIT approach, the Dengue NS1 Ag MICROLISA test was used to determine the accuracy, specificity, positive predictive value, and negative predictive value for the Dengue NS1 antigen. The assay showed an 82.76% specificity and a 92.06% sensitivity. Furthermore, there was a 95.87% positive predictive value and a 70.59% negative predictive value. (Table 4).

Table 4: Comparison of two method for the detection of NS1 Antigen by RIT (rapid Immuno-chromatography test method and NS1 antigen detection by ELISA method.

	NS1 ELISA Positive (n=126)	NS1 ELISA Negative (n=29)
NS1 RIT positive (n=121)	116	05
NS1 RIT Negative (n=34)	10	24
Sensitivity	92.06% (95% CI= 85.89% to 96.13%)
Specificity	82.76% (95% CI= 64.23% to 94.15%)
Positive Predictive Value	95.87%
Negative Predictive Value	70.59%

In the present study, we investigate the association of dengue diagnostic parameters with platelet count on the 1^st day of hospital admission. For this analysis, we considered 155 dengue positive cases that showed positivity of NS1antigen only, combined NS1 and IgM antibody, and IgM/IgG antibody. Out of these 155 cases, 46 subjects had a platelet count less than 1.0lakh/ml, and 109 subjects had a platelet count greater than 1lakh/ml. The rate of NS1 antigen positivity was significantly higher in patients (n = 21, 80.76%) with platelet count less than 1.0 lakh/ml when compared to (n = 5, 19.24%) platelet count more than 1.0lakh/ml as presented in Figure 2. When NS1 was found, thrombocytopenia was more frequently connected than when antibodies were found (P value <0.001).

Figure 2: Comparison of platelet counts with Dengue detection parameters.

DISCUSSION:

Dengue has evolved to be a serious health issue for the public across the Indian subcontinent and its surrounding nations over the past few decades²⁵. Establishing an early detection of a dengue infection within a few days of the onset of symptoms is crucial for both early and effective healthcare control of dengue epidemics as well as timely and accurate patient therapy.

In this study, we used patient data to investigate the association between age and laboratory-confirmed clinical dengue. The highest number of dengue virus affected patients were in the age group of 21–30 years, and the second most common affected age was 31–40 years. There is a correlation between age and a higher chance of developing clinical dengue¹³. Throughout the period, the percentage of dengue cases was greater in women than in men in the age range 21-30 year. A dengue virus infection can cause a wide range of symptoms, from a slight fever that goes away on its own to a severe sickness that can be fatal. A sickness known as "symptomatic dengue" is defined as a temperature exceeding 40°C, accompanied by rashes, myalgia, arthralgia, headache, nausea, vomiting, and retroorbital pain²⁶. The symptoms of rash and dyspnea demonstrated a positive association with dengue, whereas joint, bone, and muscle pain, rhinorrhea, and a confluence of nausea and diarrhea exhibited a negative relationship. Headaches and myalgia were more frequently reported in the dengue-positive group²⁷.

Dengue infection can be identified within the laboratory setting through the utilization of a diverse range of methodologies, encompassing the detection of RNA by real time RT-PCR or nested RTPCR²⁸,the examination of antigens, the evaluation of antibodies and fluorescence immunoassay (FIA)²⁹. We use an immunochromatographic card assay to examine the patterns of circulating antigen NS1, antibodies IgM and IgG during the progression of the disease in order to evaluate the combined diagnostic value of fast assays. While ELISA assay can provide a precise diagnosis of dengue infection but it is an expensive, time-consuming procedure that is out of the reach of even the majority of tertiary care hospitals³⁰. The 5-minute Rapid Card test is an easy test that may be completed without complex equipment, making it possible to administer it even in places without a lot of laboratory space²⁹.

Serum containing the highly specific NS1 antigen can be found one to nine days after the start of a fever during dengue virus infection^31,32. In clinical practice, point-of-care rapid diagnostic tests (RDTs) have been widely utilised as the primary diagnostic technique for dengue NS1 antigen and dengue-specific IgM detection³³. Dengue IgM begins increasing on day 4, following the commencement of the disease. In this investigation, blood samples from 155 dengue patients were examined, and dengue-specific IgM or IgG antibodies as well as dengue NS1 antigen were confirmed in the lab by RDT. Based on the results of the combined NS1 antigen and the IgM antibody, 69.67% (108/155) of the sera were found to be positive for DENV infection, and 5.16% of the sera tested positive for both IgM and IgG antibodies on the day of hospital admission (Day 1). Compared to RDTs that relied solely on antibody or antigen detection, these assays showed a significant increase in accuracy over the course of the patient presentation period by integrating the findings of both NS1 antigen and IgM antibody detection^34,35. Combining antigen- and antibody-based testing has definite benefits, and it should be used in reference assay and RDT formats alike.

The current investigation examined the changing patterns of NS1 antigen detection and IgG/IgM antibody kinetics in all dengue-positive patients from the first day of hospital admission to the seventh day. As hospitalization and treatment progressed, we noticed a tendency toward a decrease in NS1 antigen detection and an increase in antibody IgG/IgM detection. The results of the present investigation showed a correlation between the number of days after the beginning of fever and the sensitivity of NS1 antigen and antibody detection^36,37.

This study evaluated the dengue ELISA test and the rapid immunochromatographic test for dengue NS1 antigen detection in order to facilitate early dengue diagnosis. A total of 126 cases were positive for NS1 antigen by ELISA method, whereas 116 cases were positive by the rapid card test. Thus, only 10 cases (7.9%) of NS1 positivity were missed by the rapid immunochromatographic card test. In this study, sensitivity and specificity were 92.06% and 82.76%, respectively, when NS1 antigen was considered on rapid immunochromatographic test kits when compared to ELISA test kit^38,39. The combination of the NS1 antigen card test and ELISA assay provides the most reliable and cost-effective diagnosis of dengue^40,41. Rapid ICTs, on the other hand, have the benefit of being quick, easy to perform, and requiring minimal expertise. They can be completed in a matter of minutes. An attempt has been made to determine the relationship between thrombocytopenia and the dengue serological marker in this study. If the platelet count is less than 100,000/mL, it is considered thrombocytopenia⁴².The study revealed that NS1 positivity (80.76%) was higher in thrombocytopenia patients than IgM/IgG positivity (30.43%), indicating a strong association amongst NS1 and thrombocytopenia in contrast to antibodies IgG and IgM^43,44. NS1 antigen detection exhibited the maximum sensitivity in the early stages, although IgM detection was more sensitive in the latter stages. In terms of cost, convenience of use, and speed, NS1 RDT and ELISA are both beneficial for early dengue diagnosis.

CONCLUSION:

The current investigation demonstrated a highly substantial correlation between thrombocytopenia and dengue parameter NS1-positive cases. Determining dengue NS1 may shorten the window of opportunity within the first several days of illness. Testing for both IgM antibodies and dengue NS1 antigen enables higher diagnostic rates.

CONFLICTS OF INTEREST OF EACH AUTHOR:

The authors declare that there is no conflict of interest.

AUTHOR CONTRIBUTIONS:

Concept, Design, proof reading – HVP; Supervision & Funding – PNJ; Execution of experiment, Data collection– DA.; Literature search & Writing – LP and RZ; Analysis and/or interpretation of Data – NKS

ACKNOWLEDGMENT:

Special thanks to staff members of Rhythm Hospital, Khambhat (Gujarat) India.

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Received on 18.02.2024 Revised on 13.07.2024

Accepted on 05.11.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1515-1520.

DOI: 10.52711/0974-360X.2025.00217

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.