Validation of Small Plasma Volume to Monitor HIV-1 Viral Load by Reverse Transcriptase Enzyme Activity Assay
Reverse transcriptase enzyme activity (RT) assay is recommended to monitor viral load (VL) for HIV-infected patients in resource-limited settings by WHO, which has many advantages such as low-cost, less technical expertise requirement, no contamination issues and excellent-concordance with the gold standard nucleic acid amplification test (NAAT). Monitoring VL in pediatric patients is still a big challenge due the large plasma volume requirement of the test. Our study aims to validate whether small plasma volume is feasible to monitor HIV-1 VL in pediatric patients by RT assay.
Materials and Methods
In RT assay, a gel-separation step isolates virions from plasma components. The virions are then lysed and the lysates undergo a modified ELISA to measure the activity of reverse transcriptase enzyme. Two small plasma volume 0.2 mL [dilution factor 5 - DF5] and 0.5 mL [DF2] (top up to 1mL with HIV-1 sero-negative plasma or saline 0.9%) were compared to undiluted plasma on the linearity percentage to determine plasma volume and topping-up buffer in experiments with the known viral load plasma. The modified RT assay was used to measure VL for 420 pediatric patients in HIVCHI collaboration project between Sweden and Vietnam. All positive VL results were compared to RT-PCR by Bland-Altman test.
Results from DF5 had 95.39% linearity better than 88.68% (DF2) but DF5 was not chosen for further testing due to its instability with higher deviation (10.91 in DF5 vs. 8.02 in DF2). Results from DF2 had 88.86% linearity (top-up with HIV-1 seronegative plasma) and 96.01% (top-up with saline 0.9%), however, there was no significant difference (p=0.23) i.e. 0.5 mL saline 0.9% could be used as top-up buffer for 0.5 mL plasma. There were 25 positive VL over 420 pediatric patients detected by RT assay with 0.5mL plasma volume. The Bland-Altman comparison of results to RT-PCR (NAAT) showed that 95% limits of agreement were between -1.09 Log10 VL and 0.33 Log10 VL with mean difference -0.377 (%95CI: -0.524 to -0.229). And the Spearman’s paired correlation r2 was 0.89.
The new RT assay using only 0.5 mL volume plasma (DF2) topped up with saline 0.9% to 1 mL produces VL results with high linearity and comparable to RT-PCR. RT assay with small plasma volume is feasible to monitor HIV-1 VL for pediatric patients