Detection of Hepatitis B virus using dried blood spots and plasma separation cards

Abstract

Hepatitis B virus (HBV) infection is one of the major public health problems in the world. Testing and diagnosis are the first steps that provide access to prevention and treatment services; however, many undiagnosed infections remain in resource-limited settings. This is because the current tests require the use of plasma samples which can only be obtained with venepuncture and some people have poor veins that are difficult to draw blood from and some are afraid of venous pricks and therefore not willing to give venous blood for testing. Thus, the collection of blood by a small finger prick and spotting on dried blood spots (DBS) is an alternative method that can overcome the problems seen with venepuncture blood collection. Moreover, there is a newly developed blood collection method called plasma separation card (PSC) which has similar features as DBS but rather separates plasma from whole blood, and offers excellent sample stability and a higher volume capacity. Thus, the aim of this study was to evaluate the feasibility of DBS and PSC for the detection of HBV infection in resource-limited areas. A total of 60 EDTA donated blood samples, 30 HBV positive and 30 HBV negative were used in the study. The DetermineTM HBsAg 2 Rapid test was used to test for HBV in the samples. DBS and PSC samples were prepared from the 60 blood samples and the remainder of the samples were used to obtain plasma samples. Ten randomly selected samples were stored at different temperatures, that is, 4 °C and room temperature (RT), for 3 days to assess the stability of the DBS and PSCs. The detection of HBV from DBS, PSC and plasma samples was then evaluated using Architect i1000sr and GeneXpert assays. Viral DNA was also extracted from all the samples and the complete HBV surface gene was amplified by nested PCR. The PCR amplicons were then purified and sequenced using the BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, California, USA) on the AB 3500 xL Genetic Analyser DNA sequencer (Applied Biosystems, California, USA). DBS and PSC showed no significant change in HBsAg and HBV VL after 3 days at 4⁰C and RT compared to samples stored at -20 ⁰C. HBsAg was detected in 47 positive plasma samples, 35 positive DBS samples and 32 positive PSC samples using the Architect HBsAg assay. The sensitivity and specificity v of the DBS and PSC methods were compared with the gold standard plasma method and were determined using the area under the ROC curve (AUROC) analysis method. The DBS method was 76% sensitive and 56% specific while the PSC method was 80% sensitive and 56% specific. HBV viral load (VL) was detected in 26/47 plasma, 19/34 DBS and 22/31 PSC samples using GeneXpert. The mean difference of HBV DNA VL detected with DBS vs plasma samples was 0.5 log10 IU/ml while that of PSC vs plasma samples was 0.8 log10 IU/ml with a strong correlation of R 2 = 0.6019; p-value < 0.00 and R2 = 0.6726; p-value < 0.01 respectively. The Bland-Altman analysis plot showed great agreement when detecting HBV VL between plasma samples and card samples (DBS and PSC) for all storage conditions. DBS and PSC methods were able to detect HBsAg and HBV DNA VL even for samples stored at 4 °C and room temperature for 3 days. Some of the DBS and PSC samples were PCR amplified and compared with plasma samples. The samples were used further for phylogenetic analysis using Mega 7 software. Three genotypes (genotypes A, D and E) were detected. The study recommends the use of DBS and PSC blood colleection methods as the best alternative to the venepuncture blood collection method.