Broad access to HBV molecular diagnosis is a basic condition for starting therapy and monitoring the emergence of drug-resistant strains. However, ensuring this access is still a challenge in remote places and low-income settings1,11,16. In these cases, when the gold-standard tests are not available, the use of DBS and in-house techniques play a role in providing molecular diagnosis to hard-to-reach populations. Here, in-house qPCR was employed to detect and quantify HBV DNA in DBS samples. In addition, the applicability of DBS for sequencing purposes, as HBV genotyping and tracking mutations, was evaluate.
In this study, the detection limit of HBV in DBS by qPCR was 852.5 copies/mL (~ 250 IU/mL). Other studies using COBAS® TaqMan® HBV adapted for DBS have described detection limits of 914 IU/mL19 and 1400 IU/mL24. These discrepancies can be explained by differences between commercial and in-house methods, such as the target gene (S/Pol vs. Pre-Core/Core genes) and several automated steps.
The qPCR in DBS samples presented good agreement (86.40%), sensitivity (77.63%) and excellent specificity (100%) compared to serum samples. In-house qPCR was used as reference since this method was previously optimized and compared to commercial methods25 Some positive samples by commercial method were excluded to compare the results obtained in DBS what could impact in the estimation of sensitivity and specificity of the assay in this study and it is considered a limitation of the present work. Some studies carried out in DBS showed higher sensitivity values26,27,28,29, however most of them were carried out using commercial methods and with a smaller sample size. Moreover, satisfactory values of viral load and detection limit were observed in the repeatability and reproducibility analysis, demonstrating the reliability of the method.
Although HBV viral load was higher in serum compared to DBS, we observed good agreement between results from qPCR in serum and DBS as demonstrated by kappa value. Bland–Altman analysis demonstrated only few discordant results between qPCR in serum and DBS probably due to small differences in the performance of the assay among those samples. Other studies also demonstrated good correlation between qPCR for HBV in serum and DBS19,26,27. These results reinforce that the qPCR using DBS samples may be an economic and efficient alternative to HBV quantification in settings where serum sampling is not accessible.
Some variables were associated with qualitative and quantitative detection of HBV DNA in DBS, such as higher viral loads detected in qPCR, higher HBsAg values, HBeAg positivity and the absence of anti-HBe in the respective serum samples. Other studies have reported a greater HBV DNA detection in DBS samples with higher viral loads detected in serum19,29,30. Likewise, greater HBsAg and HBeAg detection, which in general are related to viral replication, were also associated with success in HBV detection by qPCR. In agreement, it was observed that anti-HBe reactive samples were more likely to have negative results in DBS, being related to a lower viral replication and, consequently, lower viral genome detection31.
Despite DBS is commonly used in mass serological screening, studies reporting its use for sequencing purpose are scarce and the robustness of DBS for molecular characterization is still an issue. Consequently, in low-income settings virus diversity is seldom determined, contributing to the lack of association of HBV DNA diversity and the hepatitis B phenotypes32,33,34.
In this study, we also investigated the genetic variability of HBV variants in 63 samples and evaluated the applicability of DBS for molecular epidemiology studies.
The comparison between paired serum and DBS sequences revealed great similarity, ranging from 98 to 100%. Phylogenetic analysis demonstrated that genotypes A and F were the most prevalent. Individuals infected with HBV/A showed higher values of HBsAg and lower AST titters compared to individuals infected with HBV/F (p < 0.005). Croagh et al.35 reported that HBV/A was associated to chronicity, while infection promoted by HBV/F tends to have limited progression and beneficial evolution, which may explain the difference in HBsAg levels.
Regarding subgenotypes, in both serum and DBS, HBV/A1 (57.1%) was the most prevalent, followed by HBV/F2 (23.8%), A2 (7.9%), F4 (3.2%), E (3.2%), D2, D3 and D4 (1.6% each). As have been stated, genotypes A, D and F are the most prevalent in Brazil36,37,38,39. On the other hand, genotype E is rarely detected in Brazil and when found, is often linked to the recent waves of African migration38,39. Here, genotype E was identified in two individuals from Angola living in Brazil. In agreement, phylogenetic analyses indicate that these HBV/E isolates are closely related with strains from Angola and Guinea.
It is noteworthy that HBVA1 was the most prevalent subgenotype in the Southeast, while F2 was the most frequent in Northeast. High proportion of HBV/F in Northeast was previously observed by Mello et al.36, and more recently, confirmed by Lampe et al.38. Phylogenetic analyses revealed that samples from the Northeast and Southeast had a heterogeneous distribution along the tree, suggesting that HBV strains circulating in these two regions don’t present a monophyletic origin. Brazil is a continental country where differences in colonization influenced the HBV genotype distribution along the regions39. However, expressive migration flows between the Northeast and Southeast over time may play a role in HBV dispersal and mixing of viral variants. Although this study does not suggest a distinction between the isolates from the two regions, studying the HBV variability distribution by region is crucial for increasing the knowledge of HBV dispersal patterns and for surveillance of viral variants circulating in chronic carriers.
In this study, 10 serum sequences displayed mutations in HBsAg gene. All mutations found in serum were also detected in the respective DBS sequences (when available), showing the potential of this sampling as an alternative for molecular analysis. All the amino acid changes were detected within the major hydrophilic region (MHR), where the determinant “a”, the main target of B and T cells, is located. Mutations in this region can affect the antigenicity of HBsAg and may be related to occult HBV infection and escape from vaccine-induced immunity40.
Overall, Y100C was the most frequent substitution, being present in 6/10 sequenced samples. Despite this mutation has been associated with occult HBV infection41,42,43, in this study all carriers were HBsAg-positive. As demonstrated by Mello et al.44, Y100C alone may not affect HBsAg production, secretion or HBsAg affinity by commercial serological assays, as for our samples. Thus, the presence of other potential reasons that may influence on HBsAg detection should be further investigated. In addition, L109R/V mutations were present in 2/10 samples and have been related to HBV vaccine escape and virus evasion to the host immune system7,45.
Regarding resistance mutations, the double rtM204V/I-rtL180M mutation in polymerase gene (to lamivudine, telbivudine and entecavir—partial) was observed in both serum and DBS samples from an HIV-treated patient. As observed by Komas et al.18, this finding reinforces the robustness of DBS to detect clinically relevant mutations. The accurate detection of mutations with clinical significance, as vaccine-escape and resistance mutations, is essential for better design immunization strategies and managing chronic carriers under antiviral therapy.
Our study has some limitations such as the selection of samples with viral loads above 1000 copies/mL. In addition, the assay described herein target only one region S/Pol of the HBV genome. Moreover, DBS is a less sensitive method providing a lower HBV quantification when compared to the gold-standard serum sampling, which can lead to an underestimation of viral loads. Due to this limitation, optimizations are still necessary to provide a larger number of sequenced DBS samples. DBS samples could be useful for initial screening followed by second test in serum samples before to initiate the treatment. In addition, DBS could be used to evaluate mutations and for molecular epidemiology studies when serum sampling is not available. At our knowledge, this study analysed the largest number of HBV sequences using DBS available in literature, thus contributing to increase the knowledge in this setting.
In conclusion, our results reinforce the applicability of DBS for molecular analyses, such as HBV quantification and sequencing, using in-house techniques. In low-income settings and remote locations, where the gold-standard tests are not available, the use of DBS for molecular purpose may be a convenient and economical alternative, increasing the access to HBV diagnosis. Providing alternative diagnosis methods to low-income and hard-to-reach population, who are frequently unaware of their carrier status, may close important gaps in HBV control.
