Of 82 patients with plasma HIV-1 RNA previously undetectable with use of the AMPLICOR ultrasensitive assay, 63 (77%) had detectable HIV-1 RNA levels with use of the AmpliPrep/TaqMan assay after 18 June 2007. Among the 63 patients with newly detectable HIV-1 RNA, measurements for 36 (57%) represented a >0.5-log increase, and measurements for 16 (25%) represented a >1-log increase over the detection threshold of the previous assay. This rate of HIV-1 RNA detectability among persons with previously undetectable HIV-1 RNA was higher (P < .001) than that for a group of similarly defined patients tested with only the AMPLICOR ultrasensitive assay during both the first quarter of 2006 and June 2006 (9 [10%] of 94 patients had newly detectable HIV-1 RNA during these time periods), which suggested that some of the detectable specimens tested after 18 June 2007 had increased levels of detection because of an artifact.
The hypothesis that the AmpliPrep/TaqMan assay was more sensitive near the lower limit of detection than the AMPLICOR ultrasensitive assay was tested initially. Paired plasma specimens from the Comprehensive Care Center/Vanderbilt-Meharry Center for AIDS Research Specimen Repository were available for 28 patients with discordant results (HIV-1 RNA levels, 50-1330 copies/mL in the assays performed after 18 June 2007). Both the AmpliPrep/TaqMan and AMPLICOR assays were performed on repository specimens collected during both time periods (first quarter of 2007 and after 18 June 2007) by the Vanderbilt Molecular Infectious Diseases Laboratory in August 2007 (phase I laboratory testing; ).
Figure 1 Flow charts of 2 hypotheses tested to explain artifactual high frequencies of detectable plasma HIV-1 RNA. Phase I tested the hypothesis that the new COBAS AmpliPrep/COBAS TaqMan HIV-1 RNA detection assay (Roche) was more sensitive than the ultrasensitive (more ...)
With use of the AmpliPrep/TaqMan assay, 4 (14%) of the 28 paired specimens from the first quarter and the period after 18 June 2007 each had detectable HIV-1 RNA levels. The AMPLICOR ultrasensitive assay detected HIV-1 RNA in none of the paired specimens from the first-quarter and 4 (14%) of the specimens obtained after 18 June 2007. There was no significant difference in frequency of detectable HIV-1 RNA between assays (P = .36) or in category changes (undetectable to detectable) by assay type. The AmpliPrep/TaqMan assay yielded detectable results for 8% of those specimens in which HIV-1 RNA was undetectable by use of the AMPLICOR ultrasensitive assay. Of 6 repository specimens obtained after 18 June 2007 that had HIV-1 RNA detectable by either assay, 2 had detectable HIV-1 RNA by both assays (AMPLICOR, 315 and 365 copies/mL; AmpliPrep/TaqMan, 208 and 77 copies/mL), 2 had detectable HIV-1 RNA only by the AMPLICOR assay (65 and 167 copies/mL), and 2 had detectable HIV-1 RNA only by the AmpliPrep/TaqMan assay (50 and 110 copies/mL). The minimally increased sensitivity of the newer assay did not explain the large percentage (77%) of detectable HIV-1 RNA reported from clinical testing after 18 June 2007. Furthermore, 22 (79%) of the 28 specimens that were reported to have detectable HIV-1 RNA levels in routine clinical testing with AmpliPrep/TaqMan (HIV-1 RNA levels, 50-1330 copies/mL) were not confirmed to have detectable HIV-1 RNA levels by either assay in this later quality-assurance comparison. This indicated that the increased proportion of detectable HIV-1 RNA noted after 18 June 2007 was an artifact and was not attributable to the new assay itself.
Specimen-processing methods were evaluated next. All specimens collected for clinical testing from 18 June 2007 through 17 August 2007 had been centrifuged and then shipped frozen in the same plasma preparation tube (PPT; Becton Dickinson Vacutainer System) in which blood was collected (primary PPT). Previously, plasma had been separated into another tube after centrifugation of the PPT and then shipped frozen. The repository specimens tested subsequently had been collected in separate acid citrate dextrose tubes at the same time as the specimen that was sent for initial clinical testing and then stored frozen as aliquots of separated plasma after centrifugation. Laboratory procedures for clinical testing were changed on 17 August 2007 to again require plasma removal from the centrifuged PPT and frozen storage and shipment to the national reference laboratory in a separate secondary tube. After this change, 107 (85%) of the next 126 plasma specimens from patients who had undetectable HIV-1 RNA with use of the AMPLICOR assay in the first half of 2007 had an undetectable result with use of the AmpliPrep/TaqMan assay.
We next compared each processing method with use of specimens collected prospectively from 16 patients on stable ART regimens with
2 prior undetectable HIV-1 RNA measurements. Specimens were processed 2 ways after collection in PPTs. Centrifugation and frozen plasma shipment in the primary PPT was compared with frozen plasma shipment in a secondary tube. We also tested whether concentration of virions after thawing and before performing the AMPLICOR assay might minimize the artifacts seen in specimens frozen in primary PPTs. Specimens frozen in the primary PPT from 8 (50%) of 16 patients yielded positive results when tested with the AMPLICOR without virion concentration, and 7 (44%) of 16 of these same specimens had positive results from the AMPLICOR test after virion concentration. Nine (56%) of 16 of these primary PPT specimens had positive results from the AmpliPrep/TaqMan assay (without virion concentration) (P
= .934). All 16 patients had undetectable HIV-1 RNA from clinical testing by AmpliPrep/TaqMan of plasma samples obtained concurrently and shipped in secondary tubes to the reference laboratory, with no statistically significant difference by preparation method or assay (phase II laboratory testing; ).