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

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.

The role of dried blood spot tests in the detection of hepatitis B infection: A systematic review

“…Notably, one study ( n = 1) investigated individuals occupationally exposed to BBVs, specifically hairdressers 82 . It is important to mention that six studies did not specify the population from which the samples were taken 83–88 …”

“…82 It is important to mention that six studies did not specify the population from which the samples were taken. [83][84][85][86][87][88]…”

Current tests for diagnosis of hepatitis B virus infection and immune responses of HBV-related HCC

“…HBV antigen, antibodies and DNA could be detected by EIA and PCR method in DBS and the diagnostic accuracy and precision have been confirmed by many studies ( 61 , 62 ). In 2022, the applicability of DBS for sequencing, as HBV genotyping and tracking mutations was evaluated ( 63 ). The 2017 WHO Guidelines recommended that DBS should be considered for serological and nucleic acid testing HBV and HCV technologies where laboratory detection is not available or when patients showed poor venous access ( 8 ).…”

Genetic diversity of hepatitis B virus quasispecies in different biological compartments reveals distinct genotypes

“…Previous studies have demonstrated the applicability of dried blood spot (DBS) and oral fluid samples as convenient alternatives for HBV detection and sequencing 6 , 7 . They are less invasive, easier to collect, require minimal training and may be useful for accessing HBV diversity in settings where blood collection is difficult.…”