title: Evaluation of dried blood spot samples for hepatitis C virus detection and quantification.
abstract: Dried blood spots (DBS) could be an excellent alternative for HCV diagnosis, since it is less invasive and can be stored and transported without refrigeration.
  
  
  
  The aim of this study was to optimize quantitative and qualitative methods for HCV detection in DBS.
  
  
  
  DBS and serum samples were collected from 99 subjects (59 anti-HCV/HCV RNA positive and 40 seronegative samples). Seven extraction methods and different PCR parameters were evaluated in DBS samples in the quantitative RT-PCR (qRT-PCR) developed to amplify the 5' noncoding region of HCV. A qualitative PCR for amplification of NS5B region of HCV was also valued and the nested-PCR sequenced.
  
  
  
  The qRT-PCR showed good correlation to commercial assay for HCV viral measurement in serum. To quantify HCV RNA in DBS, it was necessary to increase reverse transcriptase and cDNA concentration. HCV RNA quantification in DBS demonstrated sensitivity of 65.9%, 100% of specificity and kappa statistic of 0.65. The median viral load of DBS samples was 5.38 log10 copies/ml (minimum value=1.76 and maximum value=10.48 log10 copies/ml). HCV RNA was detected in NS5B regions and nucleotide sequences obtained in 43 serum and 11 DBS samples. The presence of the same subtype was observed in paired serum and DBS samples.
  
  
  
  In this study, it was possible to demonstrate that, despite the low sensitivity, the optimized protocol was able to determine the viral load, as well as, the infecting HCV genotype, validating the usefulness of DBS for viral load determination and molecular epidemiology studies of HCV.
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Brunna Lemos Crespo
authors: BLC
authors: Marques
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Márcia Paschoal
authors: MP
authors: do Espírito-Santo
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Vanessa Alves
authors: VA
authors: Marques
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Juliana Custódio
authors: JC
authors: Miguel
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Elisangela Ferreira
authors: EF
authors: da Silva
authors: Hepatology Division, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors: Cristiane Alves
authors: CA
authors: Villela-Nogueira
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Lia Laura
authors: LL
authors: Lewis-Ximenez
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Elisabeth
authors: E
authors: Lampe
authors: Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil. Electronic address: lvillar@ioc.fiocruz.br.
authors: Livia Melo
authors: LM
authors: Villar
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001769:Blood / Q000821:virology*; D003890:Desiccation / Q000379:methods; D003955:Diagnostic Tests, Routine / Q000379:methods*; D005260:Female; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods*; D019562:Viral Load / Q000379:methods*; D055815:Young Adult
keywords: Dried blood spot (DBS); Genotyping; Hepatitis C virus; Optimization; Quantitative methods