The use of real-time PCR to detect hepatitis C virus RNA in dried blood spots from Brazilian patients infected chronically

• Published in: Journal of virological methods 2012-01, Vol.179 (1), p.17-20
• Main Authors: Santos, Carlos, Reis, Alexanda, dos Santos, Cintia Vilhena, Damas, Cristine, Silva, Mariliza Henrique, Viana, Mônica Valverde, Ferraz, Maria Lucia, Carnauba, Dimas, El-Far, Fabiane, Serra, Fernando, Diaz, Ricardo Sobhie
• Format: Article
• Language: English
• Subjects: ambient temperature; Antiviral Agents - administration & dosage; Biological and medical sciences; blood; Blood - virology; Brazil; chronic hepatitis C; clinical trials; cost effectiveness; Desiccation; Diagnostic Errors; Dried blood spot; Drug Monitoring - methods; Fundamental and applied biological sciences. Psychology; HCV; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis C virus; Hepatitis C, Chronic - diagnosis; Hepatitis C, Chronic - drug therapy; Hepatitis C, Chronic - virology; Humans; Interferon-alpha - administration & dosage; Microbiology; Molecular Diagnostic Techniques - methods; monitoring; patients; Polyethylene Glycols - administration & dosage; qPCR; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Recombinant Proteins - administration & dosage; Ribavirin - administration & dosage; RNA; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Specimen Handling - methods; Techniques used in virology; Virology; Virology - methods; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2011.06.012

► In this study, a quantitative real-time PCR (qPCR) procedure utilizing a DBS system was developed. ► A strong concordance was observed between the presence of HCV RNA in DBS and plasma. ► DBS for HCV detection may be sufficiently sensitive and specific for use in “real world”. Collecting and transporting samples for RNA analysis can be challenging, especially in situations where financial resources are limited. In this study, a quantitative real-time PCR (qPCR) for the analysis of HCV RNA was developed and adapted for use with dried blood spot (DBS) samples. A qPCR for HCV 5′NCR, an internal control and a calibration curve were developed, and the sensitivity, specificity and dynamic range of amplification were evaluated using a panel of viruses. Plasma and DBS samples from 100 patients who had completed four weeks of Peginterferon alfa-2b+Ribavirin treatment were collected (DBS on SS903 collection cards and transported at room temperature). After 24 weeks of treatment, samples were collected from 68 of these patients. Of the 168 samples, 2 yielded false-negative results, and 4 yielded false-positive results (sensitivity was 98%, specificity was 94.3%, positive predictive value was 96.1%, and negative predictive value was 96.9%). Additionally, 2039 DBS samples from 1114 patients currently undergoing treatment for a chronic HCV infection in a clinical trial were tested. Only 10 samples out of the 2039 yielded invalid results warranting re-collection of DBS. The detection of HCV RNA in DBS can be a cost-effective strategy for HCV treatment monitoring, especially in settings where resources are limited.