Comparison of HIV-1 RNA Measurements Obtained by Using Plasma and Dried Blood Spots in the Automated Abbott Real-Time Viral Load Assay

Abstract

c Dried blood spots (DBS) may be a promising alternative specimen type to plasma for measuring the viral load (VL) in HIVinfected individuals in resource-limited settings. However, characterization of assay performance using DBS is incomplete. In this prospective study, the VL was measured in parallel using plasma and DBS specimens collected at the same time from 157 HIV-1-infected individuals. DBS were prepared by dispensing 50 l of blood onto filter paper cards and were stored desiccated at ؊20°C. Nucleic acid extraction from plasma and DBS was performed automatically using the Abbott m2000sp instrument, and the VL was measured using the RealTime HIV-1 VL assay, which has a lower limit of detection of 40 HIV RNA copies/ml. The correlation between plasma and DBS results was good (R ‫؍‬ 0.91; P < 0.001). The mean difference in the VL (DBS minus plasma) was 0.35 log copies (standard deviation [SD], 0.47 log copies). A total of 40 (26%) paired specimens had a difference of >0.5 log copy, and in 12 (7.8%) it was >1 log copy. the VL from DBS was measurable in 95.7% of specimens with a plasma VL of >2.74 log copies (550 HIV RNA copies/ml). In summary, the VL can reliably be measured using DBS with the Abbott RealTime HIV-1 assay. The estimated lower limit of detection of this automated methodology on DBS is 550 copies/ml, a threshold that may be acceptable for periodic VL monitoring in patients on antiretroviral therapy in resource-limited settings, where early detection of virologic treatment failure is often problematic. P eriodic measurement of the viral load (VL) in plasma remains a key parameter in the follow-up of HIV-infected individuals, especially those on antiretroviral therapy (ART) (21). However, reliable VL testing in plasma requires specialized facilities that often are not available in resource-poor settings. Dried blood spots (DBS) may be an interesting alternative to plasma for periodic VL testing (1,5,7,9,12,23). DBS can be easily collected and stored without being frozen or refrigerated (2,17,22). Several studies have demonstrated the feasibility of DBS as a specimen type for VL testing using different methodologies, although limitations in terms of sensitivity and stability have been noted (3,5,9,10,12,16). In addition, the recognition of distinct subtypes is an important problem in countries where HIV variability is high, and some VL assays occasionally underquantify some variants (19,24).In a previous study, we reported the results of VL testing on DBS using the Abbott mSample preparation system, a manual RNA isolation method. Although the correlation with plasma values was acceptable, the sensitivity was significantly lower on DBS (ϳ3.5 log copies) (11). Improvements in the detection limit (similar to plasma values of ϳ400 copies/ml) are important for early recognition of virological failure in patients on antiretroviral treatment. Here, we evaluate the feasibility of using an automated RNA isolation method, m2000sp, and the Abbott RealTime HIV-1 VL assay to quantify HIV-1 RNA on DBS and compar...

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

“…Second, in order to implement the WHO recommendations of task shifting and decentralization of ART to remote settings, the consequence would be that complex procedures, including drawing blood, isolation and storage of plasma samples, and cold chain shipments to qualified labs for VL testing, should be avoided. Rather, VF should be detectable on dried blood spots (DBS), a sampling alternative that is inexpensive and easy to collect and transport and has proven application for VL testing (13,14). Third, given the fact that for accurate detection of VF, a nucleic acid amplification step remains necessary and taking into consideration the realities of contamination risks in remote labs, it was decided to concentrate on a real-time PCR approach.…”

Viral Load Detection Using Dried Blood Spots in a Cohort of HIV-1-Infected Children in Uganda: Correlations with Clinical and Immunological Criteria for Treatment Failure

“…Cell-associated residual viral replication may persist even in cART-treated patients with undetectable plasma VLs (14). In our cohort, the VLs determined from DBS may have been slightly overestimated compared to the plasma VLs, particularly in the Ͻ1,000-and 1,000-to 5,000-cp/ml groups (7). The possibility of contamination of HIV-1 DNA cannot be excluded.…”

“…Although the accuracy of VL testing with DBS sampling may be affected by prolonged storage at high temperatures and humidity (5) and by variability in nucleic acid extraction and amplification results using commercial and in-house methods (6,9), most studies have reported high correlations between plasma viral loads and DBS results when plasma VLs exceed 5,000 HIV-1 RNA copies (cp)/ml (6,7,9,10). Wholeblood samples from DBS reflect the contribution of plasma plus cell-associated viral nucleic acids and may yield higher levels of HIV-1 RNA than those from plasma, particularly in the case of lower plasma VLs (7,9).…”

“…Dried blood spot (DBS) sampling is a promising tool for virological monitoring in LMIC (5)(6)(7)(8). Although the accuracy of VL testing with DBS sampling may be affected by prolonged storage at high temperatures and humidity (5) and by variability in nucleic acid extraction and amplification results using commercial and in-house methods (6,9), most studies have reported high correlations between plasma viral loads and DBS results when plasma VLs exceed 5,000 HIV-1 RNA copies (cp)/ml (6,7,9,10).…”

Leukodepletion as a Point-of-Care Method for Monitoring HIV-1 Viral Load in Whole Blood

“…Where DBS have been trialed as an alternative sample for VL monitoring, it was suspected that proviral DNA was being coamplified with viral particle RNA. One study on the feasibility of using DBS with the Abbott RealTime VL assay found that in 9% of samples with a plasma VL of Ͻ40 copies/ml, the corresponding DBS sample had a detectable nucleic acid content (17). Furthermore, this study demonstrated with a specific RT reaction that these DBS samples had undetectable RNA levels, strongly suggesting that proviral DNA was the source of reactivity.…”