Sensitivity and specificity of rapid hepatitis C antibody assays in freshly collected whole blood, plasma and serum samples: A multicentre prospective study.

BackgroundThis study evaluated performance of two hepatitis C virus (HCV) rapid diagnostic tests (RDTs) performed by intended users in resource-limited settings.MethodsTesting was conducted at three facilities in two countries (Georgia, Cambodia) using matched fingerstick whole blood, plasma and serum samples. Investigational RDTs were compared with a composite reference standard (CRS) comprised of three laboratory tests, and a reference RDT.ResultsIn matched samples from 489 HCV positive and 967 HCV negative participants, specificity with both investigational RDTs was high using either reference method (≥98.4% in all sample types). Sensitivity was lower in whole blood versus plasma and serum for both RDTs compared with the CRS (86.5-91.4% vs 97.5-98.0% and 97.3-97.1%) and reference RDT (93.6-97.8% vs 100% and 99.4%). Sensitivity improved when considering only samples with detectable HCV viral load.ConclusionSensitivity was highest in serum and plasma versus whole blood. The World Health Organization prequalification criterion (≥98%) was narrowly missed by both RDTs in serum, and one in plasma, possibly due to the intended user factor. Performance in whole blood was considered adequate, given potential roles of HCV infection history, improved sensitivity with detectable viral load and performance similarities to the reference RDT

Overview of all results per test and VLP.

<p>Numbers indicate how many input concentrations were detected per VLP and test, i.e. 3 = 50, 10 and 2 IU/ml concentrations detected, 2 = 50 and 10 IU/ml detected, 1 = 50 IU/ml detected, 0 =  VLP not detected at any concentration. The overall sensitivity for each test was calculated as number (#) of VLPs detected (D)/total number of VLPs (n = 129, i.e. # of VLPs detected + # of VLPs not detected [ND], excluding the WHO p24 standard).</p

Amino acid sequence divergence (%) of p24 in VLP panel and LANL reference subtypes after pairwise sequence alignment.

<p>Amino acid sequence divergence (%) of p24 in VLP panel and LANL reference subtypes after pairwise sequence alignment.</p

Phylogenetic relationship between Gag amino acid sequences of VLP panel members (red) and Los Alamos National Laboratory (LANL) subtype reference sequences (black).

<p>Gag subtype reference sequences were downloaded from the LANL website <a href="http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html" target="_blank">http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html</a> and filtered for subtypes present in the VLP panel. The phylogenetic tree was constructed using the neighbour joining method (Clustal W). The scale bar indicates branch length, expressed as the number of substitutions per site.</p

Detection of VLPs before and after heat-denaturation at 10 IU/ml.

<p>VLP preparations of 50 IU/ml were diluted at 1:5 in PBS and heat-denatured for 5 min at 100°C. Results for the non-heat treated VLPs for the Abbot Architect and Perkin Elmer Alliance were taken from the complete panel analysis and heat-treated VLP measurement was conducted separately. For the bioMérieux VIDAS DuoUltra, heat and non-heat treated samples were analysed in parallel. Highlighted in red are VLPs with loss of p24 detection between 1.9–3-fold for the Perkin Elmer Alliance and ≥3-fold for bioMérieux VIDAS DuoUltra and Abbott Architect.</p