Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection

Cristianne Sousa Bezerra,Moyra Machado Portilho,José Napoleão Monte Da Cruz,Ana Carolina Da Fonseca Mendonça,Lívia Melo Villar,Cristiane Cunha Frota,Carolina Pimentel De Azevedo,Jakeline Ribeiro Barbosa,Bárbara Vieira Do Lago

doi:10.1038/s41598-022-05264-1

Cristianne Sousa Bezerra, Moyra Machado Portilho + Show 7 more

Open Access

https://doi.org/10.1038/s41598-022-05264-1

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Abstract

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.

Highlights

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions
The present study aimed to evaluate the usefulness of dried blood spot (DBS) for HBV quantification and sequencing, using in-house techniques
Broad access to HBV molecular diagnosis is a basic condition for starting therapy and monitoring the emergence of drug-resistant strains

Summary

Introduction

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of serum samples can be an obstacle for molecular studies in hard-to-reach populations such as elder people, children, drug users and people living in remote places[11]. In all these cases, dried blood spot (DBS) samples may be an easy, economic and less-invasive alternative. The advent of a quantitative in-house real time PCR that can be adapted for DBS samples could improve the access to HBV molecular diagnosis in areas of limited infrastructure[16,17]. The use of DBS in molecular epidemiology studies, to better understand the role of genotypes and viral mutations in the course of hepatitis B is crucial to promote ways to access HBV diversity in settings where the gold standard methods are not available

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