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No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding “target not detected” results by CTM.
The Abbott RealTime HIV-1 assay (ART; Abbott Molecular Inc., Des Plaines, IL) and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM; Roche Molecular Systems, Inc., Branchburg, NJ) are approved by the U.S. Food and Drug Administration (FDA) for in vitro quantification of HIV-1 RNA in human plasma. ART is performed on an integrated m2000sp/m2000rt instrument system (Abbott Molecular Inc.), while CTM is performed on the COBAS AmpliPrep Instrument (CAP; Roche Molecular Systems, Inc.) and the COBAS TaqMan 48 Analyzer (CTM48; Roche Molecular Systems, Inc.) in an undocked instrument configuration or the COBAS TaqMan Analyzer (CTM96; Roche Molecular Systems, Inc.) in a docked or undocked instrument configuration. Both assays are highly automated and utilize real-time PCR methods to decrease hands-on time requirements while also reducing the potential for contamination with previously amplified reaction products. ART offers four different sample extraction volumes (0.2-, 0.5-, 0.6-, and 1.0-ml application protocols), with minimum sample input volumes ranging from 0.7 to 1.5 ml (Abbott RealTime HIV-1 Test package insert; Abbott Molecular Inc., 2007). CTM offers only a single 0.85-ml sample extraction volume protocol, which requires a sample input volume between 1.0 and 1.05 ml (COBAS AmpliPrep/COBAS TaqMan HIV-1 test package insert, rev. 2.0; Roche Molecular Systems, Inc., 2008).
Analytical sensitivities (95% detection rate) for ART range from 25 copies/ml (95% confidence interval [CI], 20 to 33 copies/ml) for the 1.0-ml application protocol to 119 copies/ml (95% CI, 102 to 150 copies/ml) for the 0.2-ml application protocol, with the corresponding lower limit of quantification (LLoQ) ranging from 40 to 150 copies/ml, respectively (Abbott RealTime HIV-1 test package insert; Abbott Molecular Inc., 2007). The analytical sensitivity of the more commonly used ART 0.6-ml application protocol is 39 copies/ml (95% CI, 33 to 49 copies/ml), with a corresponding LLoQ of 40 copies/ml (Abbott RealTime HIV-1 test package insert; Abbott Molecular Inc., 2007). For CTM, the analytical sensitivity (95% detection rate) is 40 copies/ml (95% CI, 35 to 48 copies/ml), with the LLoQ set at 40 copies/ml for the CE-marked assay and 48 copies/ml for the FDA-approved assay (Bryan Cobb, Roche Diagnostics Corp., personal communication).
Both ART and CTM are approved for the quantification of HIV-1 group M (subtypes A to H) in the United States. In addition, ART is also approved for the quantification of HIV-1 groups O and N. Although HIV-1 group M subtype B remains the predominant HIV-1 subtype circulating in the United States, recent reports suggest an increasing presence of group M non-B subtypes and non-group M strains, primarily in immigrant populations (1, 3, 11, 12).
While recent studies have generally reported good correlation between ART and CTM (13, 14), there have also been reports of significant discordance in viral load (VL) involving HIV-1 group M strains (4-7). Therefore, we conducted a study to determine if HIV-1 RNA was detectable by ART among a large number of clinical specimens submitted to our reference testing laboratory from laboratories located throughout the United States for HIV-1 load testing and previously yielding “target not detected” results by CTM. An additional aim of this study was to determine the distribution of HIV-1 genotypes and subtypes among any specimens yielding significantly discordant HIV-1 results with VLs of >200 copies/ml by ART.
Unique clinical plasma specimens submitted to our reference testing laboratory between August and December 2008 for routine HIV-1 load testing by CTM and yielding “target not detected” results were collected for possible evaluation. Only specimens with a minimum remaining volume of ≥1.6 ml were selected for inclusion in this study, and they were stored at −70°C until tested by ART. ART was performed on a single integrated m2000sp/m2000rt system using either the m2000 0.2 ml HIV-1 RNA US, version 2.00, software application or the m2000 0.6 ml HIV-1 RNA US, version 1.00, software application. CTM was performed on either of two undocked CAP/CTM96 systems using AMPLILINK 3.1.1 software. Both ART and CTM were performed according to the manufacturers' instructions, including environmental contamination checks performed periodically throughout the ART testing period as recommended by the assay manufacturer.
A total of 1,201 unique clinical specimens were tested by ART, with 943 and 258 specimens tested by the ART 0.2-ml (LLoQ = 150 copies/ml) and 0.6-ml (LLoQ = 40 copies/ml) protocols, respectively. No patient data (e.g., state of residency, treatment history, duration of infection, etc.) were available for the patients from whom these specimens were obtained. Therefore, geographic origins of the specimens were assigned according to the locations of the clinical laboratories that submitted the specimens, with the states grouped into nine geographic regions according to the National Notifiable Diseases Surveillance System operated by the Centers for Disease Control and Prevention, U.S. Department of Health and Human Services.
Among the 1,201 specimens tested, 1,190 yielded valid ART results, and these specimens originated from laboratories located in 40 states representing all nine U.S. geographic regions (Fig. (Fig.1).1). While the majority of these specimens originated from laboratories located in New England, there was representation of all nine U.S. geographic regions (Fig. (Fig.1).1). Among the 936 unique clinical specimens yielding valid results with the ART 0.2-ml protocol, 30 specimens (3.2%) contained detectable but not quantifiable HIV-1 RNA (<150 copies/ml) (Table (Table1).1). Among the 254 unique clinical specimens yielding valid results with the ART 0.6-ml protocol, 43 specimens (16.9%) yielded detectable HIV-1 RNA, including a single specimen with a measurable VL of 47 copies/ml and 42 specimens containing detectable but not quantifiable HIV-1 RNA (<40 copies/ml) (Table (Table1).1). Environmental contamination checks performed during the ART testing period showed no evidence of potentially contaminating HIV-1 sequences in the laboratory areas where ART was performed.
While clinical data were not available for those patients whose specimens yielded detectable HIV-1 RNA by ART in this study, a retrospective review of HIV-1 RNA testing previously performed in our reference testing laboratory revealed that 90% (27 of 30) and 84% (36 of 43) of these patients tested with the ART 0.2-ml and 0.6-ml protocols, respectively, had evidence of previous HIV-1 testing (quantitative and/or genotypic antiviral resistance). These findings suggested that the majority of the patients were already confirmed to be HIV-1 infected and may have been on successful highly active antiretroviral therapy (HAART) regimens with suppression of HIV-1 replication when the specimens were collected. Since suppression of HIV-1 replication during HAART would obfuscate significant differences in VL between ART and CTM, the inclusion of such specimens was a major limitation of the present study.
Although there were differences between the results of CTM (target not detected) and ART (detectable HIV-1 RNA) among some clinical specimens, the degree of VL discordance was quite small, with a maximum VL difference of 150 copies/ml. The importance of such minor differences was difficult to assess, particularly when clinical data were unavailable for review. However, other studies have reported the presence of transiently detectable but low-level HIV-1 RNA (i.e., blips) in patients undergoing HAART, especially when using highly sensitive assays, and such findings have been suggested to be of limited clinical significance (8-10).
The lack of significantly discordant VL results (difference of >200 copies/ml) among the clinical specimens included in the present study indirectly suggests that the majority of these specimens submitted for routine HIV-1 load testing in our reference testing laboratory during the study period were from patients undergoing successful HAART with suppression of HIV-1 replication and/or they did not contain atypical or non-group M HIV-1 strains. The minor differences in VL observed with some specimens included in our study were likely due to variation in specimen sampling and the inherent imprecision of these assays at low HIV-1 RNA levels, as suggested by the 95% CIs of the analytical sensitivities of these assays and as observed by other investigators (2). Despite possible underquantification of HIV-1 RNA that may be associated with atypical HIV-1 strains and an inability to detect group O HIV-1 strains by CTM (4-7), use of CTM in our laboratory did not yield false-negative results or underquantification of HIV-1 RNA that would adversely affect the management of patients from various U.S. geographic regions.
We thank Abbott Molecular Inc. for providing the instruments, reagents, and consumables used for this study.
Published ahead of print on 30 December 2009.