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Journal of Clinical Microbiology, May 2009, p. 1543-1545, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02134-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Impact of Discrepancies between the Abbott RealTime and Cobas TaqMan Assays for Quantification of Human Immunodeficiency Virus Type 1 Group M Non-B Subtypes

M. Wirden,^1,2* R. Tubiana,^2,3 F. Marguet,³ I. Leroy,¹ A. Simon,⁴ M. Bonmarchand,⁴ Z. Ait-Arkoub,¹ R. Murphy,³ A. G. Marcelin,^1,2 C. Katlama,^2,3 and V. Calvez^1,2

Department of Virology, Pitié-Salpêtriere Hospital, Paris, France,¹ Inserm U943, Paris, France,² Department of Infectious Diseases, Pitié-Salpêtriere Hospital, Paris, France,³ Department of Internal Medicine, Pitié-Salpêtrière Hospital, Paris, France⁴

Received 7 November 2008/ Returned for modification 13 January 2009/ Accepted 9 March 2009

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Viral loads in 249 clinical samples from individual patients infected with human immunodeficiency virus type 1 non-B subtypes were determined with both the Abbott RealTime and Cobas TaqMan assays. The differences exceeded 0.5 log for about 20% of samples and 1 log for 3%, with higher values always from the Abbott assay in the latter cases.

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The quantification of the plasma virus load (VL) is an essential element for monitoring human immunodeficiency virus (HIV)-infected patients in clinical practice. Various VL assays based on different technologies are available commercially (1). All are dependent on the HIV genotypic sequence, which is characterized by a high degree of variability, resulting in various groups, subtypes, and circulating recombinant forms (CRFs) (5). VL assays are typically optimized for the quantification of HIV type 1 (HIV-1) group M subtype B strains, which predominate in the United States and Europe. However, the prevalence of infection with non-B subtypes in these regions is steadily increasing, and such subtypes predominate worldwide (3, 5, 9). The reliability of VL assays in this context of high-level genetic diversity is thus critically important.

Two new HIV-1 RNA extraction-quantification systems based on real-time PCR, a cutting-edge molecular quantification technology, are now available: the Abbott RealTime (ABB) HIV-1 assay (Abbott, Wiesbaden, Germany) and the Cobas AmpliPrep/Cobas TaqMan (CTM) HIV-1 assay (Roche Diagnostics, Mannheim, Germany). These systems can be used to quantify HIV-1 group M subtypes, but recent studies have reported concerns about non-B subtypes (4, 6). Possible primer or probe mismatches in the CTM assay have also been reported (2). We assessed the impact of this problem in clinical practice by evaluating the correlation between results from the two assays and the real frequency and magnitude of the differences observed with the group M non-B subtypes in a routine clinical setting. We carried out both assays upon each new request for VL determination for samples from patients already known to be infected with one of these strains, whether the patients were on or off antiretroviral therapy, without other selection criteria. For both assays, we used the same sample that was collected for the regular virologic follow-up in the Departments of Internal Medicine and Infectious Diseases of Pitié-Salpêtrière Hospital, Paris, France, and treated in the laboratory as usual. Thus, no additional sample was necessary. Only one sample from each patient was included. To evaluate the agreement between the amounts of HIV-1 RNA detected by the two assays, we had to be sure of the presence of viruses in the samples. For this reason, we excluded all samples in which the VL was below the detection limit (1.60 log genome copies/ml) by both assays from the study. For samples with VLs quantifiable by only one assay (P1 samples), the result from the other assay was replaced with a value of 1.59 log genome copies/ml. We made this choice deliberately to represent a best-case scenario, considering that the VL was just below the detection limit. There was a risk of overestimation, but this will be discussed with the results. We compared the values obtained by the two assays by using Wilcoxon tests. We also recorded the number of discordant results with differences exceeding the clinical cutoff point of 0.5 log (7, 8, 10). Bland-Altman analyses were used to represent the degree of agreement between the results from the two assays.

Among the 621 clinical samples that have been tested, 227 plasma samples harbored VLs above the detection limit of both assays (P2 samples) and 22 were P1 samples. The samples were further assigned to two groups. The first group (hereinafter referred to as the CRF02 group) included samples (n, 113, of which 13 [11%] were P1 samples) from patients infected with a CRF02_AG strain, the main non-B subtype in France and one of the most frequently detected non-B subtypes in western Europe. The second group (hereinafter referred to as the other group) included samples (n, 136, of which 9 [7%] were P1 samples) from patients infected with any other non-B subtype. The various subtypes and other CRFs were distributed as follows in the other group, with each number indicating the number of samples for the given subtype or CRF(s): subtype A, 13; subtype C, 10; subtype D, 14; subtype F, 9; subtype G, 11; subtype H, 2; subtype J, 3; CRF01_AE, 13; CRF06_cpx, 21; CRF09_cpx, 7; CRF11_cpx, 5; CRF14_BG, 7; CRF15_01B, 2; CRF05_DF, CRF12_BF, CRF13_cpx, CRF18_cpx, and CRF19_cpx, 1 each; and nontypeable virus, 14.

For the CRF02 group, the mean VLs (and standard deviations [SD]) were 3.35 (1.25) log copies/ml by the CTM assay and 3.31 (1.35) log copies/ml by the ABB assay. Thus, the CTM assay gave slightly higher results, with a small mean difference between the results of the two assays of 0.05 log (0.40) but with a great SD and no statistically significant difference (P = 0.08). The coefficient of correlation (R²) between the results obtained in the two assays was 0.92 (Fig. 1a). The difference between the results of the two assays exceeded 0.5 log for 22 patients (19%), with values for 14 patients (including 4 with P1 samples) higher by the CTM assay and those for 8 patients (including 2 with P1 samples) higher by the ABB assay. The difference exceeded 1 log for two patients (2% of the total in the CRF02 group, including one patient with a P1 sample) among the latter eight patients.

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FIG. 1. Correlation curves and Bland-Altman analyses comparing the VL values obtained with the ABB assay and the CTM assay. (a and c) Data for the patients infected with a CRF02 subtype (n = 113). (b and d) Data for the patients infected with other HIV-1 group M non-B subtypes (n = 136).

For the other group, the mean VLs were 3.50 (1.21) log copies/ml by the CTM assay and 3.51 (1.21) log copies/ml by the ABB assay. These values were not significantly different, and R² was 0.84. However, the difference exceeded 0.5 log copies/ml for 26 patients (19%), with higher VLs for 11 patients (including 1 with a P1 sample) by the CTM assay and for 15 patients (including 1 with a P1 sample) by the ABB assay and no particular subtype or CRF identified predominantly in association with these discordances. The difference exceeded 1 log for 4 patients (3% of the total in the other group, including 1 with a P1 sample) among the 15 patients who had higher VLs by the ABB assay.

The results obtained with the two systems were strongly correlated, particularly for the CRF02 group. This situation is satisfactory, especially since these assays are based on the same underlying principle for quantification but differ in all other respects: in the extraction system, the target of primers, and probe design (the integrase gene target and detection after hybridization for the ABB assay versus the gag gene target and detection after cleavage for the CTM assay) and in the quantification method (the use of a calibration curve for the ABB assay versus that of an internal quantification standard for the CTM assay). Bland-Altman analyses showed a uniform distribution of VLs around two mean differences (ABB assay results minus CTM assay results) that were very small: –0.05 and 0.01 log copies/ml for the CRF02 and other groups, respectively (Fig. 1c and d).

However, differences between the two measurements exceeded our predetermined cutoff point of 0.5 log copies/ml for about 20% of the samples in both groups in a balanced way for the two assays. It should be noted that 0.5 log copies/ml is a particularly stringent cutoff point, as it is normally used to detect relevant variation between two values obtained with the same assay. Bland-Altman analyses showing agreement intervals (mean ± SD of 1.96) exceeding ±0.5 log bear this out (Fig. 1c and d).

We chose deliberately to represent a best-case scenario, replacing VLs below the detection limit by 1.59 for the P1 samples. Despite this optimistic position, the P1 samples accounted for 17% of samples (8 of 48) with differences exceeding 0.5 log while they accounted for only 9% of all samples (22 of 249). However, regarding these differences, the higher values for the P1 samples showed the same distribution between the two assays as those for the P2 samples. Moreover, the exclusion of the small number of patients with P1 samples did not significantly change the results (data not shown), except for the percentage of samples in the CRF02 group with significant differences (to 16% from 19%). Thus, this optimistic choice does not change the message of this study and even reinforces it.

These results confirm that, despite the strong correlation between the findings of these two systems, virologic follow-up must be based on results from the same assay, if possible. As no system is perfect, the physician should not hesitate to request VL determination to be performed with two different assays to highlight underestimation, particularly in cases of discrepancy between the VL and the CD4 count or clinical observations or for pregnant women, whatever the infecting subtype. Of course, this counsel assumes sufficient resources, but it is particularly important because, as shown here for both groups, some differences may exceed 1 log (going up to 2.6 log in this study) under the best of circumstances. For these great differences, the VLs determined by the ABB test were systematically higher. Moreover, in both Bland-Altman analyses, the few values outside the agreement intervals were consistently higher for the ABB assay (Fig. 1c and d). Thus, the version of the CTM assay available in mid-2008 seems to be unreliable in some cases. This finding is consistent with previous data (2, 4), but such large differences were found to be relatively rare (<3%) in a routine monitoring context and unrelated to a particular strain.

 
* Corresponding author. Mailing address: Laboratoire de Virologie, Hôpital Pitié-Salpêtrière, Bât. CERVI, 75013 Paris, France. Phone: 33 1 42 17 74 09. Fax: 33 1 42 17 74 11. E-mail: marc.wirden{at}psl.aphp.fr

Published ahead of print on 18 March 2009.

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Journal of Clinical Microbiology, May 2009, p. 1543-1545, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02134-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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