Validation of Cobas AmpliPrep/Cobas TaqMan HIV‐1 Test on dried blood spots

Abstract

The plasma specimen is the gold standard for viral load monitoring, the key method to assess the effect of antiviral chemotherapy and to monitor progression of the disease toward AIDS. Nevertheless, several works endorse the use of dried blood spots (DBS) on filter paper for the reliable quantification of the levels needed to take therapeutic decisions, detect of treatment failure and monitor the occurrence of drug resistance. The purpose of this study was to validate the use of Cobas AmpliPrep/Cobas TaqMan HIV‐1 test version 2.0, with DBS. To evaluate the performance of the above mentioned kit, three stages were involved: 1‐ Standardization of DBS working conditions, 2‐ Stability studies at three temperature conditions and 3‐ Performance evaluation of the kit using this alternative specimen. Additionally, the viral load was quantified in parallel (plasma and DBS) to 43 genetically characterized samples, with different levels of viral load. The Pearson correlation coefficient was calculated and the prediction of the value of RNA in plasma starting from the obtained value in DBS was made. Linear regression analysis was performed and coefficients of variation in precision assays were calculated. The best conditions pickups to the work with DBS were: 100 µL of blood (2 spots/50 µl), dried time between 16 and 18 hours at room temperature and, elution of the blood, 2 hours, between 2 and 8°C; in TRIS‐EDTA buffer. The samples on DBS proved to be stable during the study periods. A strong correlation was attained between the measurements of viral load in plasma and DBS samples (r=0.96). The detection rate was 90.7 and the coefficient of variation between the values obtained in plasma‐DBS sample pairs averaged 3.42%. The CAP/CTM HIV‐1 test provided a linear response in DBS, from 330 copies/mL to 420 000 copies/mL. Overall, coefficients of variation in precision tests were below 10%. Cobas AmpliPrep/Cobas TaqMan HIV‐1 test version 2.0 had a good performance using DBS. High detection rate was obtained with DBS specimen. Our results clearly support the use of CAP/CTM HIV‐1 test with DBS, as it reliably quantifies the levels needed for therapeutic decision‐making and detection of treatment failure.