ABSTRACT
BACKGROUND: 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. OBJECTIVES: The aim of this study was to optimize quantitative and qualitative methods for HCV detection in DBS. STUDY DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.
Subject(s)
Blood/virology , Diagnostic Tests, Routine/methods , Hepacivirus/isolation & purification , Hepatitis C/diagnosis , Hepatitis C/virology , Specimen Handling/methods , Viral Load/methods , Adolescent , Adult , Aged , Aged, 80 and over , Desiccation/methods , Female , Humans , Male , Middle Aged , Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Analysis, DNA , Young AdultABSTRACT
O objetivo deste estudo foi otimizar protocolos e padronizar métodos de diagnóstico molecular para infecção pelo HCV em amostras de sangue coletado empapel de filtro (SPF) para facilitar o acesso ao diagnóstico em áreas remotas ou com recursos limitados. Para isto, 99 indivíduos forneceram amostras pareadas de soro e SPF, dentre os quais 59eram anti-HCV reagente e 40 eram não reagentes em suas amostras de soro. As amostras anti-HCVreagentes foram submetidas à técnica de quantificação comercial. Para o desenvolvimento da RTPCRquantitativa (RT-qPCR) in house, uma curva padrão interna foi construída utilizando umplasmídeo contendo o inserto do HCV e iniciadores e sonda foram desenhados para a região 5´NCdo HCV. Para otimização da técnica, a concentração de cDNA, transcriptase reversa e númerosde ciclos de reação foram avaliados. Para a extração de RNA de HCV em amostras de SPF, setemétodos foram avaliados. A sensibilidade, especificidade, correlação, reprodutibilidade epresença de inibidores da RT-PCR quantitativa também foram determinados. O conjunto deQIAamp DNA Mini Kit foi o método mais eficiente para extração do RNA do HCV em SPF. A RTqPCRin house desenvolvida foi capaz de quantificar o HCV no soro de 44 amostras onde a medianade carga viral foi inferior aquela observada na técnica comercial (log10 4,94 e log10 6 cópias deHCV/mL, respectivamente)...
The aim of this studywas to optimize protocols and standardize molecular diagnostic methods for HCV infection in driedblood samples (DBS) to facilitate access to diagnosis in remote areas or with limited resources. Forthis, 99 individuals provided paired serum and DBS samples, where 59 of them were anti-HCVreactive and 40 were non-reactive in their serum samples. Anti-HCV positive samples were subjectedto commercial quantification technique. For the development of quantitative RT-PCR (RT-qPCR) inhouse, an internal standard curve was constructed using a plasmid containing the insert and HCVprimers and probe designed for the 5' non coding (NC) region of HCV. For optimization of thesetechniques, the concentration of cDNA, reverse transcriptase and numbers of cycles of reaction wereevaluated. For the extraction of HCV RNA in DBS samples, seven methods were evaluated. Thesensitivity, specificity, correlation, reproducibility and presence of inhibitors in quantitative RT-PCRwere also determined. QIAamp DNA Mini Kit was the most efficient method for HCV RNA extractionamong DBS samples. The in house RT-qPCR was able to quantify HCV among 44 serum sampleswhere the median viral load was lower than that observed in commercial technique (4.94 log10 and 6log10 copies of HCV / mL, respectively). The range of HCV detection using in house RT-qPCR was 10-109 copies of HCV per reaction...
