RESUMO
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.
Assuntos
Análise Mutacional de DNA , DNA Viral/genética , Teste em Amostras de Sangue Seco , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Mutação , Adulto , Estudos de Casos e Controles , Coinfecção , DNA Viral/sangue , Farmacorresistência Viral/genética , Feminino , Hepatite B/sangue , Hepatite B/virologia , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Filogenia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Carga ViralRESUMO
Dried blood spots (DBS) testing might increase the access for Hepatitis B virus (HBV) diagnosis, but little is known about the performance of these assays in real life conditions. This study aims to evaluate the diagnostic accuracy of HBsAg, anti-HBc and anti-HBs detection in DBS in clinical settings and field studies and to evaluate demographic and risk behaviour according the presence of HBsAg and anti-HBc. Paired sera and DBS samples were obtained from 2309 individuals from 3 groups, defined as follows: G1: clinical setting (n = 5-19), G2: general population (n = 1305) and G3: vulnerable individuals that could be more exposed to blood contact (n = 485). Sera and DBS were tested using commercial enzyme immunoassay (EIA), with some modifications added. Using DBS samples, the specificity values were above 90 % for HBsAg and anti-HBc in all groups and for anti-HBs range from 58.6%-85%. HBsAg testing had the best performance in GI (sensitivity = 84.4 %) and among those samples that the paired serum also presented anti-HBc marker (sensitivity = 91.6 %). High sensitivity of anti-HBc testing in DBS samples was observed in GI (80.8 %) and among HBV active cases (HBsAg+/anti-HBc+) (98.4 %). Testing of anti-HBs in DBS showed the highest sensitivity in GIII (65.5 %), in previous HBV exposed and cured individuals and when serum titers were above 100 IU/mL (86.7 %). DBS samples could be used for screening and prevalence studies for HBsAg and anti-HBc, particularly in clinical settings and among HBV active cases in field studies.
