Hepatitis B virus infection (HBV) is a major public health threat and can pose a silent transmission [1 Over a million people die each year from liver cirrhosis or hepatocellular carcinoma as a result of infection with the hepatitis B virus (HBV), which affects close to 350 million individuals worldwide. Globally, almost 45% of the inhabitants reside in regions with high chronic HBV prevalence rates [2]. HBV are highly dangerous infection for community; false negative results leave a threat of silent transmission and spreading of diseases among people [3]. Among the several viral antigens of Hepatitis B virus, HBsAg is an important viral antigen which is recognised as a superior marker for Hepatitis B virus detection. HBsAg, earlier known as Australia antigen is the first serological marker to circulate in the blood of infected individuals even 2-4 weeks before the appearance of the clinical symptoms. The levels of HBsAg are particularly elevated during the symptomatic phase and decline thereafter [4]. Hepatitis B virus infection transmits via blood/blood products, needle prick injuries, sexual relationships and vertically from mother to foetus [5]. Since, Hepatitis B virus is potentially infectious and leading to serious complications, true positives have to be identified earlier for better treatment. Early and accurate detection of Hepatitis B virus infection using sensitive and specific methods allow investigators to evaluate the status of Hepatitis B virus infection and develop strategies to prevent transmission. Hence, it is also essential to validate the detection methods prior to allowing their use in laboratories and to avoid false positive and false negative result [6]. Now-a-days many techniques are available to detect HBsAg in patients sample such as rapid ICT, ELISA, Enzyme Immunoassay (EIA), Nucleic Acid Amplification Test (NAT) and Polymerase Chain Reaction (PCR). Among these ELISA, EIA, NAT, PCR methods are costly and used in advanced laboratories. Rapid kits are cost effective and do not need technical human support [7].

A cross-sectional study was carried out from January 2024 to March 2024 in a tertiary care centre in central India.

A total of 5439 blood samples were received for HBsAg detection in the Microbiology Department, Government Medical College, Nagpur, India.   

• Inclusion Criteria: Samples received for HBsAg testing from various departments of the hospital

• Exclusion Criteria: Stored (>3 days)/contaminated samples, heat inactivated samples, samples which uses sodium azide as preservative

• Sample Processing: All 5439 blood samples were centrifuged and serum was separated. Serum of all the 5439 received blood samples were tested for HBsAg using both rapid ICT method (Mediline-SA HBsAg Combo Rapid Test, S.A. - Medline Pvt. Ltd.)  and ELISA Trustwell (HBsAg ELISA Kit, Athenese-Dx Pvt. Ltd.) method

Procedure 

Rapid Card Test process: Mediline-SA (HBsAg Combo Rapid Test, S.A. - Medline Pvt. Ltd.) The HBsAg Combo Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of 1) a burgundy-colored conjugate pad containing mouse anti-HBsAg antibody conjugated with colloidal gold (HBsAg Ab conjugates), 2)a nitrocellulose membrane strip containing a test band (T band) and a control band (C band). The T band is pre-coated with non-conjugated HBsAg antibody, and the C band is pre-coated with goat anti-mouse IgG antibody. When an adequate volume of test specimen is dispensed into the sample well of the cassette, the specimen migrates by capillary action across the test cassette. HBsAg if present in the specimen will bind to the HBsAg Ab conjugates. The immunocomplex is then captured on the membrane by the pre-coated non-conjugated HBsAg antibody, forming a burgundy-colored T band, indicating an HBsAg positive test result. Absence of the T band suggests a negative result. The test contains an internal control (C band) which should exhibit a burgundy-colored band of the immunocomplex of goat anti-mouse IgG/HBsAg Ab-gold conjugate regardless of the presence of colored T band. Otherwise, the test result is invalid and the specimen must be retested with another device.

ELISA Method

Trustwell (HBsAg ELISA Kit, Athenese-Dx Pvt. Ltd.) The Trustwell HBsAg ELISA Kit is a solid phase enzyme linked immunosorbent assay for the qualitative detection of hepatitis B virus surface antigen (HBsAg) in human serum or plasma. Trustwell HBsAg ELISA Kit is composed of two key components:

• Solid microwells pre-coated with monoclonal anti-HBsAg antibody

• Liquid conjugates composed of polyclonal anti-HBsAg conjugated with horse radish peroxidase (HRP-HBsAg conjugates)

During the assay, the test specimen and HRP-HBsAg conjugates are incubated simultaneously with the coated microwells, HBsAg, if present in the specimen, reacts to the anti-HBsAg antibody coated on the microwell surface as well as the HRP-HBsAg conjugates, forming sandwich complex conjugates. Unbounded conjugates are then removed by washing. The presence of the complexed conjugates is shown by a blue colour upon additional incubation with TMB substrate. The reaction is stopped with Stop Solution and absorbance are read using a spectrophotometer at 450/620-690 nm. 

Statistical Analysis 

A total number of 5439 serum samples were tested by using rapid card method and by ELISA method simultaneously. 

Data’s such as Optical Density of the samples/Positive Control/Negative Control/ Cut-off value and reactivity for rapid card method and ELISA were noted and entered into MS excel spread sheet and analysed.

Among the total 5439 blood samples tested by HbsAg rapid card, 112 samples were positive and the remaining 5327 were negative. 

Then the same samples when tested with ELISA, 105 samples were found to be positive and remaining 5334 were negative. 

Using ELISA as a gold standard confirmatory method. Observed data’s were made and then sensitivity, specificity, PPV, NPV and diagnostic accuracy were calculated using pre-formulated formulas.

In the present study, a comparison was done between rapid ICTs and ELISA for the screening of HBsAg. This study aims at comparing the analytical measures between rapid cards and ELISA. From this study results, it was found that for HBsAg screening, ELISA was more sensitive and more specific than the rapid card tests. In this study, the sensitivity of ICT was 99.04 % and specificity was 99.85 %. Similar to present study, Rahman M et al., has reported 53% sensitivity and 100% specificity of ICT [8]. In another study by Irwig L et al., has showed 97% sensitivity and 100% specificity [9].Kaur H et al., reported 93.4% sensitivity and 100% specificity for ICT [10]. Another study from Iran by Ansari MHK et al. shows comparable results of ICT with ELISA [11].

Table 1: Performance Metrics of Rapid Test

Table 2:  Diagnostic Performance Parameters of Rapid Test (Compared with ELISA)

This study demonstrates that in detecting HBsAg, ELISA was more sensitive than ICT. These rapid test devices can be used as a screening test for HBsAg only when the resources are limited, remote regions and peripheral health centres for screening purposes. Hepatitis B virus can pose a serious threat of silent transmission and spread among people and also create an urge for more sensitive assays as ELISA. The ultimate goal of this study was to recommend ELISA kits for the diagnosis of HBV irrespective of developmental and economical status of the area which detects positive cases correctly.

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