Use of the methods presented here resulted in a highly sensitive means of detection of HIV-1 RNA in plasma, with an average lower detection limit of 14.9 copies per ml, and in cellular specimens, with an average lower detection limit of 2.4 copies per μg of total cellular RNA. This was achieved by silica column extraction of total RNA from plasma, PBMC, or tissue specimens followed by the addition of the purified RNA to the Amplicor HIV-1 Monitor test.
Some previously reported boosted methods for measurement of the plasma virus load rely on virus concentration by high-speed centrifugation followed by Amplicor RT-PCR (7
). However, this approach does not control for the possibility of incomplete recovery of virus by the centrifugation step because the QS RNA is added after centrifugation, when the lysis buffer added at this stage protects the QS RNA from nucleases. In the silica-based Amplicor method, RNA is extracted directly from plasma with the QIAamp Viral RNA Kit (Qiagen). In this protocol the QS RNA is added before extraction, which, like the standard Amplicor test, controls for the efficiency of extraction as well as that of amplification.
The linearity of the silica-based method was similar to that for the unmodified Amplicor test, which is claimed by the manufacturer to extend from approximately 8 to 18,000 HIV-1 RNA copies per amplification. When using 0.025 ml of plasma per PCR mixture (standard Amplicor test), this represents approximately 320 to 720,000 HIV-1 RNA copies per ml of plasma. When using 0.5 ml of plasma, a range from approximately 16 to 32,000 HIV-1 RNA copies per ml can be expected. In good accordance with those expected ranges, our data for specimens from which RNA was extracted with silica show linearity in a range from 30 to 27,000 HIV-1 copies/ml and an average lower relative detection limit of 16 copies/ml. If needed, plasma virus load measurements might be boosted further. For instance, Shafer and colleagues (11
) boosted their nested HIV RT-PCR by combining centrifugation-based concentration with silica extraction. Also, the silica extraction method presented here may be developed further to accommodate larger plasma volumes. In preliminary experiments, we successfully performed silica extraction with up to 3 ml of plasma (unpublished observations).
Total cellular RNA purified with RNeasy silica columns was added to the Amplicor test to quantitate HIV-1 RNA from PBMCs and tonsil biopsy specimens. In contrast to the extraction of plasma, the extraction procedure itself is not controlled by coextraction of the QS RNA. Appropriate normalization of the input is, however, achieved by the measurement of the concentration of total RNA. In our hands, up to 2.6 μg of extracted total RNA could be added to an amplification reaction without significant inhibition, resulting in an average lower detection limit of 2.4 HIV-1 RNA copies per μg of total RNA. Addition of purified cellular RNA to the Amplicor test was previously reported by Tamalet et al. (12
), who monitored HAART by measuring HIV transcription in PBMCs and lymphoid cells. Their method differs from our protocol in the following two main ways: (i) QS RNA was added to lysed cells before extraction (as is done with the standard Amplicor extraction and the boosted method with QIAamp silica for plasma RNA), and (ii) about 5- to 10-fold less RNA (250,000 cell equivalents, or approximately 0.28 μg of total RNA [our calculations based on their data]) extracted by a guanidine-phenol-based method was added to the Amplicor test mixture.
Those investigators reported detectable HIV-1 RNA levels of less than 50 copies per 106 cells in patients receiving HAART for 8 weeks. Thus, their method appears to give results similar to those achieved by the protocol for cellular specimens reported here. However, our method offers the advantage of increasing the amount of input RNA, resulting in improved sensitivity.
The expected sensitivity and precision obtained by combining silica-based extraction with the Amplicor test were realized in clinical plasma, PBMC, and tonsil biopsy tissue specimens obtained from our ongoing study of antiretroviral therapy (EARTH study). Data for a representative patient are reported here. Detectable levels of HIV-1 RNA from PBMCs in the face of undetectable HIV-1 RNA in plasma (<20 copies/ml) attest to the sensitivity of the Amplicor RT-PCR with silica-extracted cellular specimens. The usefulness of these methods was substantiated by the following observations. In a subset of 16 patients receiving double- or triple-drug therapy, the numbers of copies of HIV-1 RNA in plasma, PBMCs, and, where available (13 patients), tonsil tissue were assessed by the methods described here during 1 year (unpublished data). That analysis revealed that the levels of HIV-1 transcripts were significantly correlated (P > 0.001) between PBMCs and plasma, PBMCs and tonsil tissue, and plasma and tonsil tissue (Spearman r values = 0.71, 0.61, and 0.62, respectively).
The relative ease of performance of these procedures with clinical specimens makes the tests well-suited for large-scale screening in the context of clinical studies, and our laboratory uses these methods routinely. Another potential advantage of measuring RNA in specimens of both cellular and cell-free origin by the same well-characterized RT-PCR method is that the levels of viral RNA in different compartments can be directly compared.
In summary, the methods described here provide highly sensitive, reproducible, and simplified tests for the measurement of HIV-1 RNA in plasma, PBMC, and frozen tissue biopsy specimens. Application to clinical research specimens may enhance the throughput and quality of the data, especially for patients in whom residual viral RNA levels are very low due to HAART.