Accurate HIV viral load measurement in primary health care settings using the cobas® plasma separation card

Abstract

Introduction: Plasma is considered the gold standard for HIV viral load (VL) testing, however its use is challenging due to the need for phlebotomy and centrifugation services, as well as cold chain for transporting to laboratories for testing. The use of Dried Blood Spot (DBS) specimen has allowed a rapid expansion of antiretroviral therapy (ART) monitoring in remote areas in many African countries, however, the VL in DBS may overestimate the copies of viral RNA result at the clinically relevant range of 1000 copies/ml, due to proviral DNA and intracellular RNA. The characteristics of the cobas® Plasma Separation Card (PSC) specimen are similar to fresh plasma (gold standard), so a better performance of HIV VL is expetected in PSC specimen and can be an alternative to DBS. This study aims to evaluate the performance of cobas® PSC for VL testing at primary health care facilities in Mozambique.

Methodology: HIV-1 infected adults on ART were enrolled consecutively in two health facilities in Mozambique, between August 2018 and October 2018. Capillary and venous cobas® PSC, DBS and fresh plasma specimens were collected from each patient. All specimens were tested for VL using CAP/CTM v2.0. Sensitivity and specificity of viral load using DBS, capillary and venous PSC specimens were estimated. Viral load obtained in fresh plasma specimen was used as reference and a threshold of 1000 copies/ml was considered for the analyses.

Results: From the total 613 patients included for the study, 2444 specimens including DBS (613), plasma (613), venous cobas® PSC (609) and capillary cobas® PSC (609) were collected and 2407 results were obtained. Sensitivity and specificity of the VL using venous cobas®PSC specimen at 1000 copies/ml threshold were 99.8% and 98.1% respectively, whereas for capillary cobas® PSC sensitivity was 99.6% and specificity was 97.2%. For DBS VL, sensitivity was 96.9% and specificity was 81.8%. Misclassification rate was more prominent in DBS specimens (5.9%), but lower in venous and capillary cobas®PSC with a rate of 0.3% and 0.7% respectively.

Conclusion: The cobas® PSC specimen has improved performance over DBS for more accurate VL testing aligned with plasma testing. The use of this specimen type can increase the rates of reliable VL results and this will improve the quality of VL monitoring of HIV-positive patients in low-income settings.

Fig 1. Overview of specimens collected and reportable results generated.

The diagram illustrates the total number of patients recruited for the study, number of samples collected for each specimen type, number of samples rejected in the laboratory due to poor quality, number of results not reported due to equipment failure and the total number of results reported for each specimen type.

Fig 2. HIV VL results for plasma, DBS, capillary and venous PSC specimens.

A scatter plot illustrating quantitative HIV viral load results generated across testing (n = 570) with the cobas^® PSC (capillary and venous collection) and DBS compared to plasma viral load testing. The vertical axis is increasing viral load (log copies/ml) and the horizontal axis is specimen number sorted by the reference method (increasing plasma viral load). The legend lists the 4 specimen types. Sorted specimen numbers 1–288 yield an undetectable VL from plasma specimen, followed by specimen numbers 289–372 yielding a VL <20 copies/ml, and specimen number 373–570 yield VL results>20 copies/ml.

Fig 3. Scatter plots of absolute difference in log copies/ml VL versus mean VL (bland-altman analysis).

The vertical axis is log difference between test and reference. The bias (Blue line), LOA (Limits Of Agreement in dotted red lines), SD (Standard Deviation in aqua error bar) are indicated on the plots. The legend also includes specimen number and concordance correlation coefficients.