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HIV-1 viremia is a marker of choice for staging, prognosis, and monitoring treatment efficiency in HIV infection. Among the commercial assays, the Amplicor HIV Monitor (Roche, Basel, Switzerland) test has the highest sensitivity for HIV-1 RNA quantitation in plasma with a detection limit of 200 copies per milliliter. To measure HIV-1 viremia below this threshold, boosted versions of the Amplicor assay were developed by adding a centrifugation step prior to RNA extraction and by decreasing dilution factors. In the boosted version, the increase in analytical sensitivity for HIV-1 RNA detection directly correlates with the input of plasma. For 1,500 µl of plasma, the sensitivity of the assay increases by a factor of 30. For routine clinical analysis, we use a boosted assay format with an input plasma volume of 500 µl and a lower detection limit of 20 copies/milliliter. Coefficients of intra- and interassay variation are similar to those reported for the standard assay (~30%). Thirteen (45%) of 29 plasma samples of HIV-infected individuals with undetectable viremia in the standard assay had detectable viremia between 20 and 200 copies/milliliter.