Seroprevalence study of dengue virus infection

Transmission

A. aegypti is the major transmitive agent of Dengue viruses (DVI) to human. The mosquito lives close contact with humans and lay eggs in fresh water containers and feed on humans rather than other vertebrates.^[3] Dengue virus infection (DVI) is related to different environmental factors (Weather and Climate), human behaviors, and load of mosquitoes in areas and susceptible human.^[4] During the past decades, dengue virus has emerged in South Asia and DF/DHF epidemics occurred in Bhutan, India, Maldives, Bangladesh, and Pakistan.^[2,5]

Symptoms

Symptoms of DF/DHF are high fever, skin rash, retro-orbital pain, myalgia, low numbers of platelets, and circulatory failure.^[5,6] DHF has higher death rate than primary DVI.^[7]

Diagnostic method

Dengue virus diagnosis methods include virus isolation and their characterization (molecular methods) and detection of antibodies and antigen (serological method).^[5]

DVI in Nepal

DVI was first reported in foreigners in Nepal.^[8,9] Larger outbreak of DF occurred in 9 districts in 2006.^[5,10] The outbreak occurred in Nepal following Indian, Pakistan, and Bhutan epidemic of DF/DHF in September–October 2006.^[10] The presence of DENV-1, DENV-2, DENV-3, and DENV-4 serotypes in the territory of Nepal indicates the chances for the epidemic of DF/DHF in the country.^[11,12]

In Nepal, diagnosis and management of dengue is based on clinical symptoms.^[12] Although the risk of infection is very high, fewer seroprevalence study has been conducted in Nepal.The molecular techniques cannot be adopted easily in country like Nepal due to high cost. Hence, the study highlights on the sensitive but cost effective test method for diagnosis of DVI in Nepal.

Objective

1. To compare the sensitivity of rapid diagnostic test (RDT) and immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of DVI in suspected cases.

2. To study the relation of DVI among different demographic groups.

Materials

1. Equipments [Table 1]

   Table 1: List of equipment used during study.

   Multi ELISA Reader Model 2010	Anthos, Austria
   Vortex shaker	Genie
   Oven	CG
   Digital camera	Canon
   Cold chamber	Diversified Biotech
   Ice box	Rush
   Autoclave	Life
   Glassware Borosil

   ELISA: Enzyme-linked immunosorbent assay, CG: Chaudhary group

2. ELISA kit (Standard Diagnostic INC., Korea)

3. RDT kit (Panbio, Australia).

Methods

The blood sample collection was done at Narayani Sub Regional Hospital, Bhawani Hospital and Research Center and Advance hospital, Birgunj during August 2023 to November 2023. Total 261 serum samples were collected from suspected cases having symptoms such as high fever, body ache retro-orbital pain, and skin rashes. Patient’s personal information and symptoms were collected by direct interview and recorded in “Dengue case details and laboratory investigation form.” The test was performed at Everest International Clinic and Research Center (EICRC), Kalanki, Kathmandu.

Ethical clearance

Written consent was taken from all suspected patients before collecting blood samples in case details and laboratory investigation forms.

Dengue case details and laboratory investigation form

I agree to provide blood samples in study titled “Seroepidemiological study of DVI in Parsha District of Nepal.” and agree to be part of study.

Sign: XXX

Date: XXX

Sample collection, storage and transport

Blood sample was collected in sterile, clean, dry test tube from suspected cases (5 mL from adult and 3 mL from children). Serum was separated in centrifuge at 3000 rpm for 5 min. The serum samples was transported to EICRC and stored at 2–8°C until tested.

Laboratory test

Detection of anti-dengue IgM by RDT

Panbio dengue kit (Panbio, Australia)

Procedure

Serum samples and RDT kit components were allowed to maintain room temperature (20–25°C) before performing the assay.

Panbio dengue kit was placed on dry and smooth surface and 10 μL serum specimen and two drops of buffer was added into the circular sample well. Test results were recorded after 15 min of adding the serum sample and buffer to the test kit [Figure 2].

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Figure 2:

Rapid diagnostic test kit.

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Interpretation of the result

One pink line “C” indicates no dengue infection. Two pink lines “C” and “M” indicates positive for IgM antibodies to dengue virus. This is indicative of a primary dengue infection.

Detection of anti-dengue IgM by IgM-capture ELISA (standard diagnostic inc, Korea)

Procedure

The ELISA kit was inserted into the strip holder. Five micro wells were labeled as controls, two wells as positive control and three wells as negative control (N). Monoclonal antibody and antigen was mixed (Positive control) and 100 μL diluted patient sample and positive controls were pipetted into their respective microwells of the assay plate. The plate was covered and incubated for 1 h at 37°C. The wells were washed 5 times with diluted wash buffer. 100 μL of diluted anti-dengue horse radish peroxidase conjugate solution was pipetted into the wells. The plate was covered and incubated for 1 h at 37°C. The wells were washed 5 times with diluted wash buffer and 100 μL of mixed tetra methyl benzidine solution was pipetted into each well. The plate was incubated at room temperature (15–30°C) for 10 min. A blue color was developed. Then, 100 μL of stop solution was pipetted into all wells and mixed well. The blue color was changed to yellow [Figure 3]. The absorbance of each well was taken within 30 min at a wave length of 450 nm with a reference filter of 620 nm using Multi ELISA Reader Model 2010 (Anthos, Austria).

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Figure 3:

Enzyme-linked immunosorbent assay kit.

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ELISA result interpretation

On basis of cutoff value, the test was interpreted as positive or negative. The sample was considered positive, if absorbance of the sample is greater than cutoff value and the sample was considered negative, if the absorbance of sample is less than cutoff value.

Cutoff value = mean absorbance of negative controls + 0.300.

Statistical analysis

The obtained data from suspected cases were analyzed using the Statistical Package for Social Sciences software (version 16.0).

RDT and IgM capture ELISA assay

1. Sensitivity: a/(a + c)*100% = 73.46%

2. Specificity: d/(b + d)*100% = 98.1%

3. Predictive value of positive test =a/(a + b)*100% = 90%

4. Predictive value of negative test = d/(c + d)*100% = 94.1%

5. Percentage of false negative = c/(a + c)*100% = 26.5%

6. Percentage of false positive = b/(b + d)*100% = 1.9%.

Age wise IgM positive case

The highest number 40 (81.6%) of positive cases was found in age group 15–50 years of age and the least; 4 (8.2%) cases were found in <15 age groups [Table 2].

Sex wise IgM positive cases

Thirty-three (67.4%) were male and 16 (32.6%) were female (male-to-female ratio were 2.06:1) [Table 3].

IgM positive among profession group

Students (6.2%) in profession group are found as highest IgM positive [Table 4].

Symptoms among IgM positive patients

Fever 49 (100%), headache 32 (65.3%), joint pain 19 (33.9, retro-orbital pain 22 (44.9%), and muscular pain 21 (42.8%) were found as the major symptoms in IgM positive cases [Table 5].