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Comparison of the COBAS TAQMAN™ HIV-1 HPS with VERSANT HIV-1 RNA 3.0 Assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes
Journal of Virological Methods 135 (2006) 223–228
Comparison of the COBAS TAQMANTM HIV-1 HPS with VERSANT HIV-1 RNA 3.0 Assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes Perp´etua Gomes a,b,∗ , Ana Carolina Palma a , Joaquim Cabanas a , Ana Abecasis a,d , Ana Patr´ıcia Carvalho a , Rainer Ziermann c , Isabel Diogo a , F´atima Gonc¸alves a , C´eu Sousa Lobo a , Ricardo Camacho a a
Laborat´orio de Virologia, Servi¸co de Imuno-Hemoterapia, Hospital Egas Moniz, Rua da Junqueria 126, 1349-019 Lisboa, Portugal b Instituto Superior de Ciˆ encias da Sa´ude Egas Moniz, Monte de Caparica, Caparica, Portugal c Bayer HealthCare, Diagnostics Division, Berkeley, CA, USA d Laboratory for Clinical and Epidemiological Virology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium Received 16 October 2005; received in revised form 20 March 2006; accepted 21 March 2006 Available online 3 May 2006
Abstract Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32 log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland–Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples. © 2006 Elsevier B.V. All rights reserved. Keywords: HIV-1; TAQMAN; bDNA; Subtypes
1. Introduction Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA levels (viral load) in plasma has an undisputed prognostic value and is extremely important for evaluating the response to antiretroviral therapy (Ahdieh et al., 2001; Mellors et al., 1996). The viral load reduction induced by antiretroviral treatment is a surrogate marker of treatment efficacy and is correlated to an improved clinical outcome (Haubrich et al., 2001; Marschner et al., 1998; Tilling et al., 2002). Use of highly active antiretroviral therapy (HAART) in the clinical practice has resulted in sharp declines of plasma HIV-1 RNA concentrations requiring ultra-sensitive assays to allow clinicians to
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Corresponding author. Tel.: +351 213650151; fax: +351 213650141. E-mail address: [email protected] (P. Gomes).
0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.018
monitor patients’ responses and to make appropriate therapeutic decisions. The development of commercial nucleic acid-based assays for the quantitation of HIV-1 RNA over the last decade represents a major advance in the clinical management and follow-up of HIV infected patients in the last decade. New technologies are being developed and the most recent have been real-time PCR-based methodologies. Quantitation by real-time PCR is based on the evaluation of the threshold cycle (Ct ) that continuously monitor the accumulation of products as it occurs. The amplified product is detected sooner when the initial viral load is higher. Real-time PCR has a greater precision in quantitation because the Ct value is observed when the PCR is still in the exponential phase, providing a more reliable measurement than conventional PCR, which measures the amplified product “at the endpoint” (Gibson et al., 1996). Comparison of viral load assays has been complicated by the lack of universal standards. Conflicting results were sometimes observed: while
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several studies have shown that quantitative measurements of HIV RNA obtained by different methods do not always agree (Antunes et al., 2003; Highbarger et al., 1999; Nolte et al., 1998; Segondy et al., 1998); others have shown that some assays such as the HIV-1 bDNA Version 3.0 (bDNA 3.0) assay and the Amplicor HIV-1 Monitor Version 1.5 (Amplicor 1.5) assay may generate comparable quantitation results (Anastassopoulou et al., 2001; Elbeik et al., 2000; Galli et al., 2005). HIV-1 is characterized by extensive genetic variability; this high degree of sequence diversity may impact the sensitivity of currently available nucleic acid-based assays. On the basis of genetic variation and phylogenetic analysis, HIV-1 has been divided into three groups (M, N, and O). Group M viruses, divided into subtypes (A, B, C, D, F, G, H, J and K)—19 circulating recombinant forms (CRFs) and multiple unique recombinant forms (URFs) (http://hivweb.lanl.gov/content/hiv-db/CRFs/CRFs.html) are the most prevalent and responsible for the global AIDS epidemic (Casado et al., 2005; Robertson et al., 2000; Thomson et al., 2005). Most viral load assays were initially targeted on HIV-1 subtype B, the predominant HIV-1 subtype in North America and Europe. However, in most European countries, infections with HIV-1 subtypes other than B are increasing, mainly due to immigration from Africa, Asia, or South America (Robertson et al., 2000; Thomson et al., 2002). Viral load assays should, therefore, accurately detect and quantify all HIV-1 group M subtypes and circulant recombinant forms (CRFs) and unique recombinant forms (URFs). Commercially available assays for the HIV-1 RNA quantitation in plasma are AMPLICOR HIV-1 Monitor Test 1.5 (Roche Molecular Systems), NucliSens HIV-1 QT (Organon Tecknika), VERSANT HIV RNA 3.0 (bDNA) (Bayer Diagnostics), Abbott LCX assay (Abbott Diagnostics), and more recently the newly developed Abbott real-time PCR COBAS TaqMan and also NucliSens EasyQ assays (Biomerieux). The impact of genetic variation on viral load quantitation has been well documented and may include underquantitation or even complete lack of detection with older versions of the currently approved assays (Roche Amplicor HIV-1 Monitor 1.0 and NASBA) (Alaeus et al., 1997; Antunes et al., 2003; Coste et al., 1996; de et al., 2005; Geelen et al., 2003; Ginocchio et al., 2003; Hill et al., 2004; Holguin et al., 1999; Nkengasong et al., 1998; Parekh et al., 1999; Stevens et al., 2005; Triques et al., 1999; Vandamme et al., 1996; Yao et al., 2005). The aim of this study was to evaluate the performance of the COBAS TaqMan 48 real-time PCR assay for detection and quantitation of different HIV-1 subtypes RNA in plasma, comparing the results to those obtained with the same samples using the VERSANT HIV-1 RNA 3.0 Assay (bDNA). The study was carried out using 104 blood samples, collected from individuals infected with different HIV-1 subtypes. 2. Materials and methods 2.1. Patient population Whole blood samples, drawn in EDTA tubes, were obtained from 104 different HIV-1 infected subjects with residence
in the centre-south region of Portugal. Plasma was separated from whole blood by centrifugation within 6 h of collection and was stored frozen at −70 ◦ C until tested. Thirty-seven (35.6%) patients were female and 67 (64.4%) patients were male. Patients’ median age was 35.16 [2–76]. The majority of the patients were under HAART. Co-infection of the above patients with HIV-2 was excluded. The samples covered a wide range of viral loads: 18 samples had viral load values below the detection limit (<50 copies/ml), 40 samples had viral loads between 50 and 9999 copies/ml, 33 samples between 10,000 and 99,999 copies/ml and 13 samples had viral load higher than 100,000 copies/ml, as evaluated by the bDNA assay. For analysis, samples were pooled in three groups: group B patients (46.15%) infected with subtype B (n = 48); group G (41.58%) patients infected with subtypes G (n = 38), AG (n = 4) and GB (n = 1); group P patients (12.87%) infected with subtypes A (n = 1), AE (n = 1), C (n = 7), F (n = 3) and UB (part of sequence was unsubtypable and other part corresponded to subtype B) (n = 1). 2.2. HIV-1 RNA assays 2.2.1. COBAS TAQMANTM HIV-1 HPS The HIV RNA for COBAS TAQMANTM HIV-1 HPS was isolated by a manual and generic preparation sample kit (High Pure System viral nucleic acid kit), based on the bonding of nucleic acid to glass fibers, in accordance with the manufacturer’s instructions. TaqMan technology uses the 5 –3 nuclease activity of thermostable Z05 DNA polymerase. It uses doublemarked fluorescent hybridization probes that bond specifically to the sample between primers. The probe contains a fluorescent marker (reporter) and another colorant that inhibits the reporter fluorescence (quencher). The degradation of the hybridization probe releases the reporter, causing an increase of fluorescence emission. The COBAS TaqMan test quantifies the amplicons during the exponential phase of amplification. The appearance of specific fluorescent signal is considered a critical threshold value (Ct ). The Ct is defined as the number of fractional cycles in which the fluorescence emitted by the sample exceeds a preset threshold (assigned level of fluorescence) and marks the beginning of an exponential growth phase of this signal. The COBAS TaqMan assay can validate quatitations between 40 and 10,000,000 copies/ml of HIV-1 RNA, using 0.5 ml of plasma. The COBAS TaqMan kit includes sufficient reagents to allow 4 runs of 12 tests, performed separately or simultaneously. The assay was performed including 21 samples and 3 controls, which must be present at every test run. 2.2.2. Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) The assay is based on branched DNA (bDNA) signal amplification technology, using synthetic oligonucleotide probes (capture probes, target probes, preamplifier probes, amplifier probes, and label probes) to generate signal amplification through hybridization. In this version (bDNA 3.0), the nonnatural bases isocytidine (isoC) and isoguanidine (isoG) are incorporated in all probes except those that hybridize to the target HIV-1 RNA (capture probes and target probes). Since
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isoC and isoG do not hybridize with natural bases, the nonspecific hybridization between probes containing these nonnatural bases is greatly reduced. The bDNA 3.0 Assay was performed according to manufacturer’s instructions, in conjunction with the semi-automated Quantiplex 340 system; it has a dynamic range of 50–500,000 HIV-1 RNA copies/ml (Collins et al., 1997) and requires 1 ml of plasma. A maximum of 84 samples plus 12 controls can be processed per plate, with the possibility of processing 2 plates simultaneously. In this assay, HIV RNA is hybridized to a series of oligonucleotide probes complementary to highly conserved regions of the HIV-1 pol gene (Collins et al., 1997). 2.3. HIV genotyping The pol gene was partially sequenced using an automatic sequencer (ABI Prism 3100, Applied Biosystems, Foster City, CA, USA) after RNA extraction, RT-PCR and amplification with marked nucleotides. Sequences were then directly subtyped using the REGA subtyping tool Version 1 (de Oliveira et al., 2005); sequences reported unassignable by the subtyping tool were aligned with group M subtypes and, when necessary, CRF’s reference sequences (Los Alamos database—http://hivweb.lanl.gov) using Clustal W Multiple Alignment (Chenna et al., 2003) and manually edited with BioEdit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html). Aligned sequences were all trimmed, to yield a 1302 bp fragment. Subtyping was performed using Simplot 3.2 (http://sray.med.som.jhmi.edu/ SCRoftware/simplot/). The sequences with bootscan values <70 were further analysed using the PHYLIP package v3.6 (http://evolution.genetics.washington.edu/phylip.html). Sequences where a recombination breakpoint was found in the Simplot analysis were cut at the recombination breakpoint and the resulting fragments analysed separately. 2.4. Statistical analysis The relationship between COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) results
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was evaluated via a Deming regression and a Bland–Altman analysis (Armitage et al., 2002; Bland and Altman, 1986). The Deming regression is similar to the well-known least squares regression except that the Deming regression allows for error in both the response and the explanatory variables. When measures from two different assays are compared, both results are subject to their own variability. If the slope of the Deming regression is close to 1, the relationship between the two assays is said to be linear. The Bland–Altman plot shows the mean log quantitations for each pair of observed results against their log differences. The mean log quantitations are a proxy for the unknown underlying concentration of the sample. If results from the two assays are randomly distributed around the mean log difference, the assay results are said to be linearly related, i.e. the relationship is constant through the tested range. If the relationship is linear, then the mean log difference between the two assay results is the expected bias between the two assays. 3. Results 3.1. Comparison of COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) The comparative performance of the COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) assays was assessed on the 104 specimens. Viral loads went through logarithmic conversion to allow direct comparison. The relationship between both assays was evaluated via a Deming regression and a Bland–Altman analysis (Armitage et al., 2002; Bland and Altman, 1986) (Fig. 1A). A positive linear correlation was observed (r2 = 0.88). The Bland–Altman plot shows the mean log quantitations for each pair of observed results against their log differences (Fig. 1B). The figure shows that there is a linear relationship between the two assays. On average the COBAS TAQMANTM HIV-1 HPS assay quantitated 0.32 logs higher than the Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) assays on average. There were three samples
Fig. 1. (A) Deming regression analysis between COBAS TAQMANTM HIV-1 HPS and Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) for all individual subtypes observed in the sample by. (B) Mean log quantitations for each pair of observed results against their log differences in all subtypes observed in the sample by Bland–Altman analysis.
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Fig. 2. (A) Deming regression between COBAS TAQMANTM HIV-1 HPS and Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) and (B) mean log quantitations for each pair of observed results against their log differences in all subtypes observed in the sample by Bland–Altman analysis in the subtypes grouped into three groups: B, G and P (other). The other category includes A, AE, C, F, UB. The G category includes AG, GB and G.
showing a difference in viral load greater than 1 log, the subtypes were G, AG and B. No obvious differences between results across the various subtypes were found. The correspondence between results was also studied among the previously defined three groups: Fig. 2 shows the Deming and Bland–Altman plots by [pooled] subtype. The relationship between the two assays is similar for all subtypes showing a slope lower than 1 for the Deming regression and lower than 0 for the Bland–Altman plots. The only Deming slope that is significantly different from 1 is the slope for subtype B (Fig. 3). This is due to the larger sample size for subtype B. VERSANT HIV-1 RNA 3.0 Assay (bDNA) average quantitation is lower than COBAS TAQMANTM HIV1 HPS for all subtypes but given the small sample sizes none of these differences are significantly different from 0 (Fig. 4).
Fig. 4. Mean log differences by group subtype.
4. Discussion
Fig. 3. Deming slopes by group subtype.
The comparison of HIV-1 RNA data generated with different methods remains a critical issue, since patients may be monitored by more than one assay over the course of their treatment. Earlier studies indicated that not all assays yield comparable results (Hodara et al., 1998; Lin et al., 1998; Nolte et al., 1998; Segondy et al., 1998). The observed discordance between the results of different assays or generations of assays has led to the recommendation that patients should be monitored with the same assay (Hodara et al., 1998; Segondy et al., 1998). In the present study, the COBAS TAQMANTM HIV-1 HPS quantitative assay was for the first time compared with other ultra-sensitive test – VERSANT HIV-1 RNA 3.0 – using plasma samples from 104 individuals infected with different HIV-1 subtypes. A posi-
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tive linear correlation (r2 = 0.88) between COBAS TAQMANTM HIV-1 HPS and VERSANT bDNA 3.0 was found. The regression data further suggest that COBAS TAQMANTM HIV-1 HPS yields slightly higher results (0.32 log) than VERSANT bDNA 3.0. However, the relationship between the two assays was similar across all subtypes. Compared to the VERSANT 3.0 Assay, the real-time PCR has a greater dynamic range (40–10,000,000 copies/ml) and only needs 0.5 ml plasma whereas the VERSANT assay requires 1 ml of plasma. When monitoring children, this smaller sample volume requirement might be an advantage for the COBAS assay. COBAS TaqMan 48 allows to process 48 samples per run in a 5-h period. However, due to the reduced hands-on time, the processing of large sample numbers (168 patient results per run) is easier with the VERSANT 3.0 Assay. 5. Conclusions The COBAS TaqMan 48 analyser and VERSANT 3.0 (bDNA) have excellent correlation in samples with various subtypes. Based on the fact that no significant differences were observed in these two assays concerning viral load measurements, it may be assumed that HIV-1 subtypes are equally quantitated by both assays. In conclusion, despite of reduced sample size, the observed overall concordance between viral load determinations by COBAS TAQMANTM HIV-1 HPS and VERSANT bDNA 3.0 may allow for the interchangeable use of these assays in the clinical management of HIV-1-infected subjects. Both tests are suited for viral load quantitation of genetically diverse populations of HIV-1 group M. References Ahdieh, G.L., Silverberg, M.J., Palacio, H., Minkoff, H., Anastos, K., Young, M.A., Nowicki, M., Kovacs, A., Cohen, M., Munoz, A., 2001. Discontinuation of potent antiretroviral therapy: predictive value of and impact on CD4 cell counts and HIV RNA levels. AIDS 15 (16), 2101–2108. Alaeus, A., Lidman, K., Sonnerborg, A., Albert, J., 1997. Subtype-specific problems with quantification of plasma HIV-1 RNA. AIDS 11 (7), 859–865. Anastassopoulou, C.G., Touloumi, G., Katsoulidou, A., Hatzitheodorou, H., Pappa, M., Paraskevis, D., Lazanas, M., Gargalianos, P., Hatzakis, A., 2001. Comparative evaluation of the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays and the AMPLICOR HIV-1 MONITOR v1.5 test for the quantitation of human immunodeficiency virus type 1 RNA in plasma. J. Virol. Meth. 91 (1), 67–74. Antunes, R., Figueiredo, S., Bartolo, I., Pinheiro, M., Rosado, L., Soares, I., Lourenco, H., Taveira, N., 2003. Evaluation of the clinical sensitivities of three viral load assays with plasma samples from a pediatric population predominantly infected with human immunodeficiency virus type 1 subtype G and BG recombinant forms. J. Clin. Microbiol. 41 (7), 3361–3367. Armitage, P., Berry, G., Matthews, J.N.S., 2002. Statistical methods in medical. Blackwell Science, Oxford, Malden, MA. Bland, J.M., Altman, D.G., 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1 (8476), 307–310. Casado, G., Thomson, M.M., Sierra, M., Najera, R., 2005. Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19 cpx) in Cuba. J. Acquir. Immune Defic. Syndr. 40, 532–537. Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby, J., Higgins, Desmond, G., Thompson, Julie, D., 2003. Multiple
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Comparison of the COBAS TAQMANTM HIV-1 HPS with VERSANT HIV-1 RNA 3.0 Assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes Perp´etua Gomes a,b,∗ , Ana Carolina Palma a , Joaquim Cabanas a , Ana Abecasis a,d , Ana Patr´ıcia Carvalho a , Rainer Ziermann c , Isabel Diogo a , F´atima Gonc¸alves a , C´eu Sousa Lobo a , Ricardo Camacho a a
Laborat´orio de Virologia, Servi¸co de Imuno-Hemoterapia, Hospital Egas Moniz, Rua da Junqueria 126, 1349-019 Lisboa, Portugal b Instituto Superior de Ciˆ encias da Sa´ude Egas Moniz, Monte de Caparica, Caparica, Portugal c Bayer HealthCare, Diagnostics Division, Berkeley, CA, USA d Laboratory for Clinical and Epidemiological Virology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium Received 16 October 2005; received in revised form 20 March 2006; accepted 21 March 2006 Available online 3 May 2006
Abstract Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32 log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland–Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples. © 2006 Elsevier B.V. All rights reserved. Keywords: HIV-1; TAQMAN; bDNA; Subtypes
1. Introduction Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA levels (viral load) in plasma has an undisputed prognostic value and is extremely important for evaluating the response to antiretroviral therapy (Ahdieh et al., 2001; Mellors et al., 1996). The viral load reduction induced by antiretroviral treatment is a surrogate marker of treatment efficacy and is correlated to an improved clinical outcome (Haubrich et al., 2001; Marschner et al., 1998; Tilling et al., 2002). Use of highly active antiretroviral therapy (HAART) in the clinical practice has resulted in sharp declines of plasma HIV-1 RNA concentrations requiring ultra-sensitive assays to allow clinicians to
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0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.018
monitor patients’ responses and to make appropriate therapeutic decisions. The development of commercial nucleic acid-based assays for the quantitation of HIV-1 RNA over the last decade represents a major advance in the clinical management and follow-up of HIV infected patients in the last decade. New technologies are being developed and the most recent have been real-time PCR-based methodologies. Quantitation by real-time PCR is based on the evaluation of the threshold cycle (Ct ) that continuously monitor the accumulation of products as it occurs. The amplified product is detected sooner when the initial viral load is higher. Real-time PCR has a greater precision in quantitation because the Ct value is observed when the PCR is still in the exponential phase, providing a more reliable measurement than conventional PCR, which measures the amplified product “at the endpoint” (Gibson et al., 1996). Comparison of viral load assays has been complicated by the lack of universal standards. Conflicting results were sometimes observed: while
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several studies have shown that quantitative measurements of HIV RNA obtained by different methods do not always agree (Antunes et al., 2003; Highbarger et al., 1999; Nolte et al., 1998; Segondy et al., 1998); others have shown that some assays such as the HIV-1 bDNA Version 3.0 (bDNA 3.0) assay and the Amplicor HIV-1 Monitor Version 1.5 (Amplicor 1.5) assay may generate comparable quantitation results (Anastassopoulou et al., 2001; Elbeik et al., 2000; Galli et al., 2005). HIV-1 is characterized by extensive genetic variability; this high degree of sequence diversity may impact the sensitivity of currently available nucleic acid-based assays. On the basis of genetic variation and phylogenetic analysis, HIV-1 has been divided into three groups (M, N, and O). Group M viruses, divided into subtypes (A, B, C, D, F, G, H, J and K)—19 circulating recombinant forms (CRFs) and multiple unique recombinant forms (URFs) (http://hivweb.lanl.gov/content/hiv-db/CRFs/CRFs.html) are the most prevalent and responsible for the global AIDS epidemic (Casado et al., 2005; Robertson et al., 2000; Thomson et al., 2005). Most viral load assays were initially targeted on HIV-1 subtype B, the predominant HIV-1 subtype in North America and Europe. However, in most European countries, infections with HIV-1 subtypes other than B are increasing, mainly due to immigration from Africa, Asia, or South America (Robertson et al., 2000; Thomson et al., 2002). Viral load assays should, therefore, accurately detect and quantify all HIV-1 group M subtypes and circulant recombinant forms (CRFs) and unique recombinant forms (URFs). Commercially available assays for the HIV-1 RNA quantitation in plasma are AMPLICOR HIV-1 Monitor Test 1.5 (Roche Molecular Systems), NucliSens HIV-1 QT (Organon Tecknika), VERSANT HIV RNA 3.0 (bDNA) (Bayer Diagnostics), Abbott LCX assay (Abbott Diagnostics), and more recently the newly developed Abbott real-time PCR COBAS TaqMan and also NucliSens EasyQ assays (Biomerieux). The impact of genetic variation on viral load quantitation has been well documented and may include underquantitation or even complete lack of detection with older versions of the currently approved assays (Roche Amplicor HIV-1 Monitor 1.0 and NASBA) (Alaeus et al., 1997; Antunes et al., 2003; Coste et al., 1996; de et al., 2005; Geelen et al., 2003; Ginocchio et al., 2003; Hill et al., 2004; Holguin et al., 1999; Nkengasong et al., 1998; Parekh et al., 1999; Stevens et al., 2005; Triques et al., 1999; Vandamme et al., 1996; Yao et al., 2005). The aim of this study was to evaluate the performance of the COBAS TaqMan 48 real-time PCR assay for detection and quantitation of different HIV-1 subtypes RNA in plasma, comparing the results to those obtained with the same samples using the VERSANT HIV-1 RNA 3.0 Assay (bDNA). The study was carried out using 104 blood samples, collected from individuals infected with different HIV-1 subtypes. 2. Materials and methods 2.1. Patient population Whole blood samples, drawn in EDTA tubes, were obtained from 104 different HIV-1 infected subjects with residence
in the centre-south region of Portugal. Plasma was separated from whole blood by centrifugation within 6 h of collection and was stored frozen at −70 ◦ C until tested. Thirty-seven (35.6%) patients were female and 67 (64.4%) patients were male. Patients’ median age was 35.16 [2–76]. The majority of the patients were under HAART. Co-infection of the above patients with HIV-2 was excluded. The samples covered a wide range of viral loads: 18 samples had viral load values below the detection limit (<50 copies/ml), 40 samples had viral loads between 50 and 9999 copies/ml, 33 samples between 10,000 and 99,999 copies/ml and 13 samples had viral load higher than 100,000 copies/ml, as evaluated by the bDNA assay. For analysis, samples were pooled in three groups: group B patients (46.15%) infected with subtype B (n = 48); group G (41.58%) patients infected with subtypes G (n = 38), AG (n = 4) and GB (n = 1); group P patients (12.87%) infected with subtypes A (n = 1), AE (n = 1), C (n = 7), F (n = 3) and UB (part of sequence was unsubtypable and other part corresponded to subtype B) (n = 1). 2.2. HIV-1 RNA assays 2.2.1. COBAS TAQMANTM HIV-1 HPS The HIV RNA for COBAS TAQMANTM HIV-1 HPS was isolated by a manual and generic preparation sample kit (High Pure System viral nucleic acid kit), based on the bonding of nucleic acid to glass fibers, in accordance with the manufacturer’s instructions. TaqMan technology uses the 5 –3 nuclease activity of thermostable Z05 DNA polymerase. It uses doublemarked fluorescent hybridization probes that bond specifically to the sample between primers. The probe contains a fluorescent marker (reporter) and another colorant that inhibits the reporter fluorescence (quencher). The degradation of the hybridization probe releases the reporter, causing an increase of fluorescence emission. The COBAS TaqMan test quantifies the amplicons during the exponential phase of amplification. The appearance of specific fluorescent signal is considered a critical threshold value (Ct ). The Ct is defined as the number of fractional cycles in which the fluorescence emitted by the sample exceeds a preset threshold (assigned level of fluorescence) and marks the beginning of an exponential growth phase of this signal. The COBAS TaqMan assay can validate quatitations between 40 and 10,000,000 copies/ml of HIV-1 RNA, using 0.5 ml of plasma. The COBAS TaqMan kit includes sufficient reagents to allow 4 runs of 12 tests, performed separately or simultaneously. The assay was performed including 21 samples and 3 controls, which must be present at every test run. 2.2.2. Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) The assay is based on branched DNA (bDNA) signal amplification technology, using synthetic oligonucleotide probes (capture probes, target probes, preamplifier probes, amplifier probes, and label probes) to generate signal amplification through hybridization. In this version (bDNA 3.0), the nonnatural bases isocytidine (isoC) and isoguanidine (isoG) are incorporated in all probes except those that hybridize to the target HIV-1 RNA (capture probes and target probes). Since
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isoC and isoG do not hybridize with natural bases, the nonspecific hybridization between probes containing these nonnatural bases is greatly reduced. The bDNA 3.0 Assay was performed according to manufacturer’s instructions, in conjunction with the semi-automated Quantiplex 340 system; it has a dynamic range of 50–500,000 HIV-1 RNA copies/ml (Collins et al., 1997) and requires 1 ml of plasma. A maximum of 84 samples plus 12 controls can be processed per plate, with the possibility of processing 2 plates simultaneously. In this assay, HIV RNA is hybridized to a series of oligonucleotide probes complementary to highly conserved regions of the HIV-1 pol gene (Collins et al., 1997). 2.3. HIV genotyping The pol gene was partially sequenced using an automatic sequencer (ABI Prism 3100, Applied Biosystems, Foster City, CA, USA) after RNA extraction, RT-PCR and amplification with marked nucleotides. Sequences were then directly subtyped using the REGA subtyping tool Version 1 (de Oliveira et al., 2005); sequences reported unassignable by the subtyping tool were aligned with group M subtypes and, when necessary, CRF’s reference sequences (Los Alamos database—http://hivweb.lanl.gov) using Clustal W Multiple Alignment (Chenna et al., 2003) and manually edited with BioEdit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html). Aligned sequences were all trimmed, to yield a 1302 bp fragment. Subtyping was performed using Simplot 3.2 (http://sray.med.som.jhmi.edu/ SCRoftware/simplot/). The sequences with bootscan values <70 were further analysed using the PHYLIP package v3.6 (http://evolution.genetics.washington.edu/phylip.html). Sequences where a recombination breakpoint was found in the Simplot analysis were cut at the recombination breakpoint and the resulting fragments analysed separately. 2.4. Statistical analysis The relationship between COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) results
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was evaluated via a Deming regression and a Bland–Altman analysis (Armitage et al., 2002; Bland and Altman, 1986). The Deming regression is similar to the well-known least squares regression except that the Deming regression allows for error in both the response and the explanatory variables. When measures from two different assays are compared, both results are subject to their own variability. If the slope of the Deming regression is close to 1, the relationship between the two assays is said to be linear. The Bland–Altman plot shows the mean log quantitations for each pair of observed results against their log differences. The mean log quantitations are a proxy for the unknown underlying concentration of the sample. If results from the two assays are randomly distributed around the mean log difference, the assay results are said to be linearly related, i.e. the relationship is constant through the tested range. If the relationship is linear, then the mean log difference between the two assay results is the expected bias between the two assays. 3. Results 3.1. Comparison of COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) The comparative performance of the COBAS TAQMANTM HIV-1 HPS with Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) assays was assessed on the 104 specimens. Viral loads went through logarithmic conversion to allow direct comparison. The relationship between both assays was evaluated via a Deming regression and a Bland–Altman analysis (Armitage et al., 2002; Bland and Altman, 1986) (Fig. 1A). A positive linear correlation was observed (r2 = 0.88). The Bland–Altman plot shows the mean log quantitations for each pair of observed results against their log differences (Fig. 1B). The figure shows that there is a linear relationship between the two assays. On average the COBAS TAQMANTM HIV-1 HPS assay quantitated 0.32 logs higher than the Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) assays on average. There were three samples
Fig. 1. (A) Deming regression analysis between COBAS TAQMANTM HIV-1 HPS and Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) for all individual subtypes observed in the sample by. (B) Mean log quantitations for each pair of observed results against their log differences in all subtypes observed in the sample by Bland–Altman analysis.
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Fig. 2. (A) Deming regression between COBAS TAQMANTM HIV-1 HPS and Bayer VERSANT HIV-1 RNA 3.0 Assay (bDNA) and (B) mean log quantitations for each pair of observed results against their log differences in all subtypes observed in the sample by Bland–Altman analysis in the subtypes grouped into three groups: B, G and P (other). The other category includes A, AE, C, F, UB. The G category includes AG, GB and G.
showing a difference in viral load greater than 1 log, the subtypes were G, AG and B. No obvious differences between results across the various subtypes were found. The correspondence between results was also studied among the previously defined three groups: Fig. 2 shows the Deming and Bland–Altman plots by [pooled] subtype. The relationship between the two assays is similar for all subtypes showing a slope lower than 1 for the Deming regression and lower than 0 for the Bland–Altman plots. The only Deming slope that is significantly different from 1 is the slope for subtype B (Fig. 3). This is due to the larger sample size for subtype B. VERSANT HIV-1 RNA 3.0 Assay (bDNA) average quantitation is lower than COBAS TAQMANTM HIV1 HPS for all subtypes but given the small sample sizes none of these differences are significantly different from 0 (Fig. 4).
Fig. 4. Mean log differences by group subtype.
4. Discussion
Fig. 3. Deming slopes by group subtype.
The comparison of HIV-1 RNA data generated with different methods remains a critical issue, since patients may be monitored by more than one assay over the course of their treatment. Earlier studies indicated that not all assays yield comparable results (Hodara et al., 1998; Lin et al., 1998; Nolte et al., 1998; Segondy et al., 1998). The observed discordance between the results of different assays or generations of assays has led to the recommendation that patients should be monitored with the same assay (Hodara et al., 1998; Segondy et al., 1998). In the present study, the COBAS TAQMANTM HIV-1 HPS quantitative assay was for the first time compared with other ultra-sensitive test – VERSANT HIV-1 RNA 3.0 – using plasma samples from 104 individuals infected with different HIV-1 subtypes. A posi-
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tive linear correlation (r2 = 0.88) between COBAS TAQMANTM HIV-1 HPS and VERSANT bDNA 3.0 was found. The regression data further suggest that COBAS TAQMANTM HIV-1 HPS yields slightly higher results (0.32 log) than VERSANT bDNA 3.0. However, the relationship between the two assays was similar across all subtypes. Compared to the VERSANT 3.0 Assay, the real-time PCR has a greater dynamic range (40–10,000,000 copies/ml) and only needs 0.5 ml plasma whereas the VERSANT assay requires 1 ml of plasma. When monitoring children, this smaller sample volume requirement might be an advantage for the COBAS assay. COBAS TaqMan 48 allows to process 48 samples per run in a 5-h period. However, due to the reduced hands-on time, the processing of large sample numbers (168 patient results per run) is easier with the VERSANT 3.0 Assay. 5. Conclusions The COBAS TaqMan 48 analyser and VERSANT 3.0 (bDNA) have excellent correlation in samples with various subtypes. Based on the fact that no significant differences were observed in these two assays concerning viral load measurements, it may be assumed that HIV-1 subtypes are equally quantitated by both assays. In conclusion, despite of reduced sample size, the observed overall concordance between viral load determinations by COBAS TAQMANTM HIV-1 HPS and VERSANT bDNA 3.0 may allow for the interchangeable use of these assays in the clinical management of HIV-1-infected subjects. Both tests are suited for viral load quantitation of genetically diverse populations of HIV-1 group M. References Ahdieh, G.L., Silverberg, M.J., Palacio, H., Minkoff, H., Anastos, K., Young, M.A., Nowicki, M., Kovacs, A., Cohen, M., Munoz, A., 2001. Discontinuation of potent antiretroviral therapy: predictive value of and impact on CD4 cell counts and HIV RNA levels. AIDS 15 (16), 2101–2108. Alaeus, A., Lidman, K., Sonnerborg, A., Albert, J., 1997. Subtype-specific problems with quantification of plasma HIV-1 RNA. AIDS 11 (7), 859–865. Anastassopoulou, C.G., Touloumi, G., Katsoulidou, A., Hatzitheodorou, H., Pappa, M., Paraskevis, D., Lazanas, M., Gargalianos, P., Hatzakis, A., 2001. Comparative evaluation of the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays and the AMPLICOR HIV-1 MONITOR v1.5 test for the quantitation of human immunodeficiency virus type 1 RNA in plasma. J. Virol. Meth. 91 (1), 67–74. Antunes, R., Figueiredo, S., Bartolo, I., Pinheiro, M., Rosado, L., Soares, I., Lourenco, H., Taveira, N., 2003. Evaluation of the clinical sensitivities of three viral load assays with plasma samples from a pediatric population predominantly infected with human immunodeficiency virus type 1 subtype G and BG recombinant forms. J. Clin. Microbiol. 41 (7), 3361–3367. Armitage, P., Berry, G., Matthews, J.N.S., 2002. Statistical methods in medical. Blackwell Science, Oxford, Malden, MA. Bland, J.M., Altman, D.G., 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1 (8476), 307–310. Casado, G., Thomson, M.M., Sierra, M., Najera, R., 2005. Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19 cpx) in Cuba. J. Acquir. Immune Defic. Syndr. 40, 532–537. Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby, J., Higgins, Desmond, G., Thompson, Julie, D., 2003. Multiple
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