WHO/BS/2018.2346 A Collaborative Study to Evaluate the Proposed 1st WHO International Standard for Human Adenovirus (HAdV) DNA for Nucleic Acid Amplification Techniques (NAT)

Overview

This report describes the collaborative study evaluation of candidates for the proposed 1st WHO International Standard for HAdV DNA for NAT-based assays. The choice of source material and formulation for the candidates was guided by the results of a pilot study conducted in 2016. Two lyophilized candidates were prepared, each comprising laboratory-grown HAdV type 2 diluted in 10mM Tris HCl buffer (pH 7.4), containing 1% Trehalose and 0.5% human serum albumin. Thirty-two laboratories from 13 countries evaluated the suitability of each candidate using their routine HAdV NAT-based assay. Candidates 16/306 (sample A) and 16/324 (sample B) were evaluated alongside three laboratory-grown viruses (samples C-E) and three clinical isolates comprising different HAdV types. Participants were asked to dilute samples A-E in the matrix relevant to the extraction protocol used with their HAdV assay (either plasma, MEM (for stool extraction) or whole blood (all provided)). A wide range of HAdV NAT assays were used in the evaluation, the majority of which were quantitative real-time PCR assays targeting the hexon gene. Individual laboratory mean estimates for samples A-E differed by ~4 log10 ‘copies/mL’ (SD of overall mean estimates between 0.70 and 0.91 log10 ‘copies/mL’). The variability of HAdV DNA measurements was lowest when samples A-E were diluted in plasma and highest when diluted in MEM for stool extraction. A similar pattern of inter-laboratory variability was observed for clinical isolates F-H (evaluated in quantitative assays only). For all samples interlaboratory variation was significantly greater than intra-laboratory variation. For the cultured virus samples A-E, harmonization of HAdV DNA measurements was observed when the mean estimates were expressed relative to candidates A and B. The greatest harmonization was observed when the samples were matched by HAdV type or species. For the clinical isolates, there was also some improvement in the agreement between laboratories when the mean estimates were expressed relative to candidates A and B. The results obtained from ongoing accelerated thermal degradation studies at 1 year indicate that both candidates are stable and suitable for long-term use. The results of the study indicate the suitability of both candidates A and B to serve as reference materials to help standardize HAdV NAT assays. As the type 2 virus in sample B is widely used as a common reference strainit is proposed that this candidate (NIBSC code 16/324) is established as the 1st WHO International Standard for HAdV DNA for NAT with an assigned potency of 2×10 8 IU/mL (~8.3 log10 IU/mL) when reconstituted in 1 mL of nuclease-free water.