Bastian Neesgaard , Morten Ruhwald , Henrik B. Krarup , Nina Weis [1,4]
Affiliates:  Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, 2650 Hvidovre, Denmark.  Department of Infectious disease Immunology; Section of Human Immunology; Statens Serum Institute, 2300 Copenhagen S, Denmark.  Section of Molecular Diagnostics, Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, Denmark.  Department of Clinical Medicine; Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen Ø, Denmark.
With the introduction af the new direct acting antivirals with short treatment spans and high rates of sustained virological response, treatment of hard-to-reach populations such as intravenous drug users, prison inmates, immigrants and sex workers among others is now feasible. However, a major challenge to overcome is the small percentage of hepatitis C virus (HCV) infected individuals known to healthcare systems. Therefore, there is a real need for new cost-effective and reliable test methods, as an alternative to standard venous blood testing, if diagnosis rate is to increase, and the WHOs goals for eliminating hepatitis C reached. Dried bloodspots (DBS) could represent one such method. The primary objectives of this study were to examine if anti-HCV antibodies could be detected- and HCV-RNA quantified from DBS samples, sent by regular mail. In addition, HCV-RNA value from these samples were compared to the HCV-RNA values in paired serum samples.
Forty chronic HCV infected, HIV-, and Hepatitis B negative patients, without IV drug use, who had not received antiviral- or immunosuppressant treatment within six months, were consecutively enrolled from the out-patient clinic at the infectious medicine ward, Copenhagen university hospital, Hvidovre. At baseline and after six months, dried bloodspots were prepared from blood collected in a 4 mL. standard venous puncture tube, dried for 4-6 hours on a lab bench, then stored in gas-impermeable plastic bags with a desiccator, placed in another gas-impermeable plastic bag before being sent by regular mail. On arrival, samples were frozen at -80 OC until time of analysis. At each visit a paired venous sample was performed and analyzed for HCV-RNA and Anti-HCV
Eleven patiens were lost to follow up, and three samples were faulty, leaving 66 paired serum and filter paper samples available for HCV-RNA analysis. We found a significant correlation between log HCV-RNA koncentration found in plasma, and log HCV-RNA deviated from filter paper (P < 0.0001, persons R 0.6788, R2 0.4607) figure 1. HCV-RNA derived from dried blood spots samples were consequently lower than the corresponding serum concentration, reflected by Bland-Altman bias 3.041 with SD of bias ± 0.6472. We had 67 pair DBS and serum samples available for anti-HCV determination. Of these 67 samples, anti-HCV were found in 66 of the DBS samples, with only one sample testing false negative.
HCV-RNA can be quantified from dried blood spot samples, dried at room temperature and sent by regular mail, with a significant correlation to serum concentrations. The concentrations are however lower than serum values. In our population of HCV-RNA positive patients, 98.5 % were found to have anti-HCV in blood derived from filter paper, with only one false negative sample. Dried blood spots could be a cost-effective, easy-to-use and reliable alternative to standard tests for the diagnosis of HCV infections as well as level of viremia, improving diagnostics of difficult to reach populations.