Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Application of sequential serological testing strategy for detection of Hepatitis B surface antigen (HBsAg) for diagnosing HBV infection Aseem Kumar Tiwari ⁎ , Swati Pabbi, Geet Aggarwal, Dinesh Arora, Gunjan Bhardwaj, Divya Setya, Jyoti Sharma Medanta - The Medicity Hospital, Department of Transfusion Medicine, Sector-38, Gurgaon, India ARTICLE INFO Keywords: HBsAg Testing strategy Sequential testing Sequential serological testing HBV infection HBsAg neutralization Diagnostic accuracy Reflex testing PPV ABSTRACT The diagnostic accuracy of any serological test for detection of HBsAg is not 100%. We hypothesized that the sequential testing strategy proven for anti-HIV laboratory diagnosis should also apply to other infectious disease markers like HBsAg. Therefore, we evaluated the diagnostic accuracy of these strategies, I (single test), II (two tests in sequence), III (three tests in sequence) for diagnosis in patients and blood donors and compared it to the confirmatory test for HBsAg (Neutralization Test). Samples were initially tested for HBsAg by A1- Enhanced Chemiluminescent Immuno Assay (ECLIA). Initial reactive (aliquoted donor/patient) samples were reflexly tested by A2- Enzyme Linked Fluorescence Assay (ELFA) and A3- Immuno Chromatography Assay (ICA) assays. Confirmatory neutralization assay was performed on all initial reactive samples. Four strategies (I, II A, II B, and III) that were used in this analysis were; I = A1, IIA = A1 + A2, IIB = A1 + A3, and III = A1 + A2 + A3. The results of all four strategies were compared to Gold Standard (Neutralization Test). A total of, 112, 011 blood samples (75,111 patient samples and 36,900 whole blood donor samples) were initially tested for HBsAg by A-1 (CLIA). Amongst the tested samples, 1,296, 1,188, 1,078, 1,074 samples were found to be reactive by strategy I, IIA, IIB, III respectively. We observed that the PPV (Positive Predictive Value) of Strategy III > Strategy IIB > Strategy IIA > Strategy I. Sequential serological testing strategy comprising of initial sensitive test fol- lowed by more specific test increases the diagnostic accuracy of test report as compared to a single test. Hepatitis B infection is a major public health problem worldwide. According to the World Health Organization (WHO), around 248 mil- lion individuals are chronically infected with hepatitis B virus (HBV) in the world; particularly in low- and middle-income countries and four million acute infections are reported every year. Approximately 686,000 people die each year due to complications related to chronic HBV infection (WHO, 2017). Due to limited access to diagnosis and treatment of hepatitis B, in many resource constrained settings, infected individuals are not aware of their status and present with complications in advanced stages of disease. Testing and diagnosis of HBV infection is important for prevention and treatment of hepatitis. The most com- monly performed diagnostic test to detect Hepatitis B infection is HBs Ag. Different methods for detection of HBsAg are available since the demonstration of its relation toviral hepatitis (Blumberg, 2000). Rou- tinely, serum HBsAg can be detected by enzyme linked immunosorbent assay (ELISA), immunochromatographic rapid diagnostic test, electro- chemiluminescent immunoassay, and chemiluminescent microparticle immunoassay (Amini et al., 2017; Randrianirina et al., 2008). Since no single test is 100% sensitive and specific, sequential testing strategies comprising of two or more tests to improve diagnostic accuracy have been reported. One of these reports from WHO has defined and clas- sified the testing strategies for HIV into three strategies, namely I, II and III [WHO, HIV Guidelines]. While strategy I may be adequate for blood screening, it is sub-optimal for making diagnosis in a patient or recal- ling a blood donor for post-donation counselling and follow-up. We hypothesized that the sequential testing strategy proven for anti- HIV laboratory diagnosis is applicable to other infectious disease mar- kers such as HBsAg. Therefore, we evaluated the diagnostic accuracy of these strategies, I (single test), II (two tests in sequence), III (three tests in sequence) for diagnosis in patients and blood donors and compared it to the confirmatory test for HBsAg, the neutralization test). Several laboratories continue to use strategy I, especially in blood screening. However, when it comes to disease surveillance and diagnosis, la- boratories use two or three tests. This study was a prospective analysis of test results by different serological assays used for detection of HBsAg in patients and blood donors, performed from March 2016 to July 2017 in the department of https://doi.org/10.1016/j.jviromet.2019.113726 Received 26 April 2019; Received in revised form 19 August 2019; Accepted 31 August 2019 ⁎ Corresponding author. E-mail address: draseemtiwari@gmail.com (A.K. Tiwari). Journal of Virological Methods 274 (2019) 113726 Available online 31 August 2019 0166-0934/ © 2019 Elsevier B.V. All rights reserved. T