Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis

Abstract

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

Fig 1

Bland-Altman analysis of HIV-1 load quantification between A-226 (A) or M-TFN (B) and W-903 filter papers. Nucleic acid was extracted from DBS specimens by using the NucliSENS EasyMag automated system, and NucliSENS HIV-1 v2.0 RUO kits were used to determine the HIV-1 load using the NucliSENS EasyQ analyzer. Twenty-four specimens with a detectable viral load (VL) (defined as a VL of ≥2.90 log₁₀ copies/ml) with all three types of filter paper were included in the analyses. The solid line represents the mean difference between W-903 and A-226 (−0.034 ± 0.246 log₁₀ copies/ml) or W-903 and M-TFN (−0.028 ± 0.186 log₁₀ copies/ml) filter papers, and the dotted lines represent the 95% limits of agreement (mean difference ± 1.96 standard deviations) for A-226 (−0.517 and 0.449; width = 0.965) and M-TFN (−0.393 and 0.336; width = 0.728) filter papers.