Background:  Greater value is being placed on the determination of virological failure in patients on ART. Viral load assays are performed in centralized laboratories because they require expensive equipment and technical skill. Access to viral load monitoring in resource-limited settings is hampered by transport difficulties that prevent blood samples from reaching centralized facilities rapidly. Dried blood spot (DBS) samples facilitate centralized testing since centrifugation and rapid, cold chain transportation is not required. This study aims to optimize and evaluate the performance of the BioMerieux EasyQ HIV viral load assay on dried plasma spot (DPS) and DBS.

Method:  EDTA blood samples from 150 HIV-infected patients undergoing routine viral load monitoring at the Johannesburg Hospital, South Africa were used to prepare DBS and DPS on Whatman 903 Guthrie cards. For each patient, a DBS and DPS (~50 µL) was excised and placed in 2-mL Lysis Buffer overnight. HIV-1 RNA was extracted using the EasyMAG. NASBA amplification and detection was performed using NucliSens EasyQ assay as per manufacturer's instructions. Negative and positive controls were included in each run. To determine the lower limit of detection, 5-fold serial dilutions (n = 10) of known HIV-1 concentration for DPS and DBS were made. Stability studies to evaluate the effects of longer term storage (at intervals from 72 hours to 3 months) under different conditions (–20, 4, room temperature (±28) and 40ºC) for both DPS and DBS are ongoing. Plasma viral load results were used as the standard against which DPS and DBS viral load results were compared.

Results:  Bland-Altman analysis comparing plasma viral load results to DPS and DBS viral load results showed excellent correlation with mean differences of 0.09 and 0.24 log, respectively, for viral load >3.5 logs. Of the 28 plasma samples (19%) with a viral load <3.5 logs, 16 and 24 samples were undetectable on DPS and DBS, respectively. The serial dilution results confirmed this lower limit of detection. No decline in viral load at 72 hours or at 1 week at the 4 different temperatures was detected except after 1 week at 40ºC.

Conclusions:  Reliable detection of viral load >3.5 logs is useful in improving care according to current guidelines for changing ART in resource-limited settings. The stability of the viral load results without cold storage for at least 1 week is encouraging for conditions in resource-limited settings. DBS samples are simpler to collect than DPS and provide a feasible option for improving access to HIV viral load monitoring in resource-limited settings.