Validation of the Roche MagNA Pure CompactTM Automated Extraction and Cobas TaqmanTM 48 Real-Time PCR Protocol for HIV-1 Quantitative Viral Load Measurements in Semen

Erasmus, Smit1, Shaz Ahmad1, Jonathan Ball2, Trudy Mason1, Steve Wilson1, Sarah Drury3, Geraldine Hartshorne3, Gerry Gilleran4, Steve Taylor4

1. Public Health Laboratory, HPA Birmingham, Heartlands Hospital, Birmingham, UK
2. The Institute of Infection, Immunity and Inflammation, The University of Nottingham, Nottingham, UK
3. Centre for Reproductive Medicine, University Hospital, Coventry, UK
4. Dept of GU Medicine, Heartlands Hospital, Birmingham, UK

Background

The risk of transmission of HIV from a male to his sexual partner is related to the amount of virus in his semen. None of the commercial HIV-1 viral load assays are licensed for samples other than blood plasma and all current methods for seminal plasma viral loads are based on adaptation and validation of commercial assays. We developed and validated the performance of an easy to use, small volume, single sample automated extraction method as the front end of a normal HIV-1 viral load assay. This method will enable processing of small numbers of semen samples at a time and assay concurrently with normal blood plasma HIV-1 viral load testing.

Methods

MagNA Pure CompactTM extraction followed by the Roche COBAS TaqmanTM HIV detection was optimised for input, elution and QS volumes. Various dilution panels were made by spiking known quantities of HIV-1 into HIV negative seminal plasma. Specificity was evaluated by repeat testing of HIV negative seminal plasma. The limit of detection (LOD) was determined by testing 10, 15 and 15 spiked seminal plasmas with an expected viral load of 400, 200 and 100 copies/ml. Finally a comparison with a previous published method (TNAI COBAS Ampliprep/Cobas TaqmanTM) was performed. Correlation coefficient, scatter graphs and Bland Altmanns comparisons were used in the statistical analysis.

Results

No inhibition was observed in any of the specimens tested. There was good linearity and correlation (R2=0.97) between the expected and observed values for the dilution series. The reproducibility was within acceptable limits with the highest coefficient of variation (0.35) observed at the LOD of the assay. The LOD was at least 100 copies/ml and the assay specificity was 100%.  There was a good comparison (R2= 0.87) between the MagNA Pure Compact and TNAI extraction methods. Bland Altmanns plots showed that Compact gave on average 0.31 of log10 copies lower reading.

Conclusions

The MagNA Pure Compact is a single sample automated extraction method which can be used as the front end of the COBAS TaqmanTM HIV-1 quantitative PCR in seminal plasma.