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. 1999 May;37(5):1260-4.
doi: 10.1128/JCM.37.5.1260-1264.1999.

Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues

M Fischer  1 , W Huber, A Kallivroussis, P Ott, M Opravil, R Lüthy, R Weber, R W Cone
Affiliations

Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues

M Fischer et al. J Clin Microbiol. 1999 May.

Abstract

Precise and sensitive quantitation of viral RNA in specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals has become an indispensable tool for the monitoring of the efficacy of highly active antiretroviral combination therapy. The present report describes reproducible and efficient protocols with enhanced sensitivity for quantitation of HIV-1 RNA from plasma, peripheral blood mononuclear cells, and tissues with Qiagen silica columns for RNA purification combined with the Roche Amplicor HIV-1 Monitor test for quantitative reverse transcription-PCR (RT-PCR). Extraction of RNA from 0.5 ml of plasma resulted in the detection of fewer than 20 HIV RNA copies/ml of plasma, equivalent to the centrifugation-based boosted RT-PCR assay. Silica extraction of cellular RNA resulted in the detection of fewer than 3 HIV-1 RNA copies/microg of total RNA. These techniques facilitate direct comparisons of viral loads between liquid and cellular specimens. Application of these sensitive methods may improve the assessment of the response to new antiretroviral regimens.

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Figures

FIG. 1
FIG. 1
Comparison of plasma viral load determination by using QIAamp extraction versus the standard Amplicor protocol. Aliquots of clinically obtained HIV-1-positive plasma specimens were thawed and measured by the standard Amplicor test. Other aliquots of these specimens were thawed, diluted 10-fold in HIV-negative control plasma, and extracted by the QIAamp procedure, and the purified RNA was measured by the Amplicor test. Expected viral loads were calculated as 10% of the value measured for the undiluted aliquot by the standard Amplicor test. Closed squares, data points for specimens with a detectable measurement in the QIAamp test. One diluted specimen (open square) was negative for HIV RNA (expected readout, 73 copies/ml; measured value, <17 copies/ml). Linear regression (r2 = 0.91; n = 22), as shown by the solid line, and the 95% confidence intervals (dotted lines) result in an equation of y = (0.97 ± 0.07)x − (0.14 ± 0.18) (the slope and the y-intercept values are indicated as the mean ± SE).
FIG. 2
FIG. 2
Cell mixing experiment to assess the linearity of HIV-1 RNA measurement in cellular specimens. RNA was extracted from frozen cell pellets representing mixtures of HIV-infected cells (8E5) and uninfected cells (H9). Specimens had been mixed to contain 2, 10, 100, or 1,000 8E5 cells per 106 H9 cells and 106 cells per pellet (n = 8; four duplicate measurements). Aliquots containing 297 to 409 ng of total RNA were measured by PCR. Linear regression analysis (r2 = 0.91; n = 8) as shown by the solid line and the 95% confidence intervals (dotted lines) results in an equation of y = (0.90 ± 0.11)x + (3.01 ± 0.11) (the slope and the y-intercept values are indicated as mean ± SE).
FIG. 3
FIG. 3
Kinetics of HIV-1 RNA levels in plasma and PBMCs during antiretroviral treatment. An antiretroviral agent-naive, asymptomatic, HIV-infected individual started AZT-3TC-ritonavir therapy at time zero. Plasma virus loads (number of copies per milliliter) are shown for the standard Amplicor test (circles) and the silica-based (QIAamp) Amplicor test (diamonds). Levels of cellular HIV-1 RNA (number of copies per microgram of RNA) are shown for PBMCs (triangles) and tonsil tissue (squares). Filled symbols signify detectable viral RNA, whereas open symbols indicate lower detection limits for specimens devoid of measurable HIV-1 RNA.
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