Use of dried spots of whole blood, plasma, and mother's milk collected on filter paper for measurement of human immunodeficiency virus type 1 burden

Abstract

We studied the use of dried spots of bodily fluids (plasma, whole blood, and mother's milk) on filter paper as a means of sample collection and storage for human immunodeficiency virus type 1 (HIV-1) viral load testing under stringent field conditions. Plasma placed directly in lysis buffer, which is customarily used for viral load assays, was used for comparison in all our experiments. Utilizing reconstruction experiments, we demonstrate no statistical differences between viral loads determined for plasma and mother's milk spotted on filter paper and those for the same fluids placed directly in lysis buffer. We found that the addition of whole blood directly to lysis buffer was unreliable and could not be considered a feasible option. However, viral load measurements for whole blood spotted onto filter paper correlated with plasma viral load values for both filter spots and lysis buffer (Pearson correlation coefficients, 0.7706 and 0.8155, respectively). In conclusion, dried spots of plasma, whole blood, or mother's milk provide a feasible means for the collection, storage, and shipment of samples for subsequent viral load measurement and monitoring. Virus material spotted and dried on filter paper is a good inexpensive alternative for collecting patient material to monitor the HIV-1 viral load. Measuring the HIV-1 burden from whole blood dried on filter paper provides a suitable alternative for low-technology settings with limited access to refrigeration, as can be found in sub-Saharan Africa.

FIG. 1.

Correlation between results for filter spots and fluids placed in lysis buffer. Log₁₀ measurement of the HIV-1 RNA content in plasma or milk as assessed by the LTR-based, nucleic acid sequence-based amplification (Retina-Rainbow) assay. The virus-containing fluid was dissolved directly in lysis buffer or was initially spotted on filter paper (four spots of 50 μl each) prior to RNA isolation and quantification. The values for the spotted samples are plotted against the values for the corresponding samples dissolved directly in lysis buffer. The linear correlation between the two formulations, together with the 95% confidence interval, is shown. The broken line with the open circles is human plasma-containing virus from a panel of cultured isolates, encompassing subtypes A, B, and CRF01. The solid line with the closed circles is human milk-containing virus from the same panel of cultured isolates.

FIG. 2.

(A) HIV-1 subtype B-infected patient sera assessed by the Retina-Rainbow (LTR-based nucleic acid sequence-based amplification) assay. Two hundred milliliters of the sera was dissolved in lysis buffer or dried as spots (four spots of 50 μl each) on filter paper prior to RNA isolation and quantification. The log₁₀ RNA level values for the spotted sample were plotted against the corresponding value for the sample dissolved in lysis buffer. The solid lines represent the linear correlation between the two formulations, and the dotted lines represent the 95% confidence interval. (B) Log₁₀ RNA level values for the panel of patient samples as measured by the Retina-Rainbow assay (x axis) plotted against the log₁₀ RNA values as determined by the Amplicor 1.5 assay.

FIG. 3.

Sera infected with the HIV-1 non-B subtype was assessed by the Retina Rainbow assay as described in the legend for Fig. 2A. The closed symbols represent 19 subjects sampled in The Netherlands infected with HIV-1 subtypes A, C, D, K, CRF01, CRF02, and CRF06. The open symbols represent 19 HIV-1 subtype C-infected subjects sampled in Ethiopia.

FIG. 4.

Assessment of whole blood collected as dried spots on filter paper for the monitoring of HIV-1 viral burden. (A) The log₁₀ RNA values derived from 200 μl blood spotted on filter paper were plotted against the log₁₀ RNA values derived from 200 μl plasma dissolved in lysis buffer (solid line/closed circles) or against the log₁₀ RNA values derived from 200 μl plasma spotted on filter paper (broken line/open circles). (B) A pairwise comparison of the log₁₀ RNA values from whole blood spotted on paper to the log₁₀ RNA values from plasma dissolved in lysis buffer, which is the standard formulation.