Implementation of an HIV-1 Triple-Target NAT Assay in the Routine Screening at Three German Red Cross Blood Centres

Abstract

Background: Blood product safety was significantly improved by the introduction of NAT testing in the late 1990s, resulting in a strong decrease of transfusion-transmitted infections (TTIs). Due to the occurrence of HIV-1 NAT test failures as a consequence of mismatch mutations in the amplicon regions of mono-target NAT assays, the Paul Ehrlich Institute mandated the implementation of multi-target NAT assays for HIV-1 in 2014. Commercial suppliers mostly developed dual-target NAT assays, with only one implementing a triple-target NAT assay.

Methods: The HIV-1 triple-target NAT assay v3 (GFE Blut) was tested on mutated specimens and synthetic DNA bearing mutations that resulted in sample underquantification or false-negative test results. In addition, data from 2 years routine testing at three German Red Cross Blood centres were analysed.

Results: The HIV-1 triple-target PCR could compensate for all mutations tested and could compensate the loss of one amplicon without a significant loss of sensitivity. Data from 2 years routine testing showed a solid performance.

Conclusion: The HIV-1 triple-target v3 assay (GFE Blut) can compensate mutations in target sequences better than a dual-target assay and is applicable to high-throughput screening, thus increasing blood product safety.

Keywords: Blood safety; HIV-1 triple-target NAT; HIV-1 variants; High throughput testing; Multi-target PCR; NAT.

Fig. 1

A Phylogenetic (maximum likelihood) tree of an alignment of the gap-pol-env genomic region (nt 790-8795 on reference clone HXB2 (K03455)) of the case 4 and 5 isolate sequences and HIV-1 reference sequences. B Subtree of (A) of the subtype B clade with bootstrap values. Phylogenetic analysis was done using maximum likelihood (ML) statistics with MEGA6 [13] using default settings. As a basis for phylogenetic analysis a prebuild HIV-1 reference alignment (HIV1_REF_2010_genome_DNA) from the HIV-1 database (Los Alamos, NM; www.hiv.lanl.gov) was downloaded and the two sequences of case 4 and 5 isolates, as well the sequence of the WHO international standard (KJ019215) were manually integrated and the alignment further manually edited using Jalview 2 (www.jalview.org [14]). To show robustness of the phylogenetic analysis, the trees were calculated using bootstrapping with 100 replicates and the analysis was repeatedly done with different parts of the alignment (e.g. the gag, pol, env ORF regions alone or with a part of the LTR region) using different statistical methods (UPGMA, neighbour joining, maximum likelihood). Insertions and variable regions were removed manually from the alignments before phylogenetic analysis was performed.

Fig. 2

A 95% limit of detection for the single amplicons. The described subtypes are represented by gBlocks. B Ct values for the single amplicons obtained from gBlocks for a concentration of 1,000 cop/rxn.