COMPARISON OF HEPATITIS QUANTITATIVE AND QUALITATIVE METHODS USING VIVEST SAMPLE TRANSPORTATION MATRIX VS FROZEN PLASMA

 COMPARISONOF HEPATITIS QUANTITATIVE AND QUALITATIVE METHODS USING VIVEST SAMPLE TRANSPORTATION MATRIX VS FROZEN PLASMA

R.M. Lloyd Jr¹, R. Diaz², C. Loveday³, K. T. Presley⁴, R. L. Mathis⁴, P. M. Feorino⁴, Z. Grossman⁵, and M. Holodniy⁶

¹ViveBio, LLC, Buford, GA, USA, ²Retrovirology Laboratory, Federal University of São Paulo, São Paulo, Brazil, ³ICVC Charitable Trust HQ, Buckinghamshire, UK, ⁴Research Think Tank, Inc. Buford, GA, USA, ⁵Clinical Virology Laboratory and National Reference Laboratory, Sheba Medical Center, Ramat Gan, Israel, and ⁶AIDS Research Center, VA Palo Alto Healthcare System, Palo Alto, CA, USA

 

Background:Several diseases of the liver, collectively known as hepatitis, are caused primarily by five viral agents of which have been reasonably well characterized. This group of hepatitis virus comes from a wide range of viral families. Hepatitis A virus is a picornavirus, a small single strand RNA virus; hepatitis B virus (HBV) belongs to the hepadnavirus, family of double stranded DNA viruses; hepatitis C virus (HCV) is a flavivirus, a positive, single-strandedRNA virus; hepatitis E, also an RNA virus, is similar to a calicivirus. Hepatitis D that is also known as “Delta” virus (HDV) is a single-stranded circular RNA that completely relies on HBV co-infection. In all, these viruses make up > 300 million world wide infections and an effective solution for logistics, specifically safe cost effective transportation is needed for testing.

Objective:This study presents an evaluation of a dried, 1 mL plasma transport device, ViveST™ (ST), for dried plasma samples containing three different hepatitis virus; HBV, HCV and HDV analyzed by high complexity assays used in research and routine patient management.

Methods: Frozen and dried 1 mL plasma samples containing HCV, HBV or HBV plus Hepatitis D co-infection were compared using TRUGENE® HCV 5’NC Genotyping, TRUGENE® HBV Genotyping kits (Siemens Diagnostics) and/or a novel Hepatitis D specific qPCR Real-Time quantitative virus load (log₁₀ RNA copy/mL) assay (Centro de Genomas, São Paulo, Brazil). Specimens were extracted using QIAamp Viral RNA Mini Kit or bioMerieux Nucleic Acid Extractor utilizing BOOM methodology. Extracted viral RNA or DNA from frozen or dried (ST) plasma was used in a single-step RT-PCR or PCR reaction for each of the two-genotyping assays as recommended by manufacturers product insert. Analysis of TRUGENE sequences was accomplished using the integrated TRUGENE software and MuTanker™ (ViveBio, LLC). Sequence between frozen and dried specimen were directly compared.

Results:Regardless of Hepatitis virus nucleic acid type, RNA or DNA, the ST dried specimens were stable. The overall success rate of dried plasma amplification was 100% and the mean accuracy of nucleic acids comparisons was >99%. There was no difference in RNA vs. DNA stability over the 14 days of combined transit and storage for frozen to dried comparisons (genotyping) or RNA stability of Hepatitis RNA in 7 days. ST dried plasma qPCR results were concordant with frozen plasma and one HDV specimen amplified from dried specimen but was unable to amplify from ST dried specimen.

Conclusion:  These data and other studies show the effectiveness of ST dried ambient transport of both RNA and DNA virus for complex analyte testing regardless of viral family type or structure. Furthermore, the ST dried specimen was shown to mirror real world transport conditions with comparable results to the standard of the industry, frozen plasma specimen.