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Journal of Clinical Microbiology, August 2005, p. 4234-4236, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4234-4236.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improved Sensitivity of Human Immunodeficiency Virus Type 2 Subtype B Plasma Viral Load Assay

Florence Damond,1,2* Gilles Collin,1,2 Diane Descamps,1,2 Sophie Matheron,1 Sophie Pueyo,3 Audrey Taieb,3 Pauline Campa,4 Antoine Benard,3 Geneviève Chêne,3 and Francoise Brun-Vezinet1,2

Hôpital Bichat Claude Bernard, Paris, France,1 INSERM U552, Paris, France,2 INSERM U330, Bordeaux, France,3 Hôpital Saint Antoine, Paris, France4

Received 27 January 2005/ Accepted 23 April 2005


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ABSTRACT
 
We developed a new assay for human immunodeficiency virus type 2 plasma RNA quantification based on a previous format. The new version performed significantly better than the original regarding the detection of subtype B, allowing the detection of 14 out of 36 plasma RNAs in the subtype B-infected patients not detected with the original version.


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TEXT
 
Human immunodeficiency virus type 2 (HIV-2) differs from HIV-1 by its lower pathogenicity and lower plasma viral load (3, 12, 13). Two longitudinal studies of patients living in West Africa reported a link between HIV-2 viral load and the CD4 cell count decline and death (2, 11). In the French Agence Nationale de Recherche sur le SIDA HIV-2 cohort study, we confirmed the very slow progression of HIV-2 disease and showed that, as in HIV-1 infection, plasma RNA quantification was predictive of the risk of clinical progression (14).

Another major characteristic of HIV-2 is its high degree of genetic diversity. At least eight HIV-2 subtypes have been described previously (4, 8, 10, 15), and the two major subtypes, A and B, have been identified in France (6). In the absence of an approved commercial assay for HIV-2 RNA quantification, we developed an assay based on a real-time PCR in 2001 (7). The frequency of plasma RNA positivity did not depend on the subtype, but the mean plasma viral load was slightly higher in subtype A infection than in subtype B infection (7). This apparent difference in sensitivity was confirmed by longitudinal studies. Here we developed an improved version of this assay and compared the results with those obtained with the previous format.

We tested EDTA plasma samples stored at –80°C collected from patients included in the French Agence Nationale de Recherche sur le SIDA national cohort, who originate from various African countries and are infected by HIV-2 subtype A (n = 20), subtype B (n = 36), or subtype H (n = 1), as determined by pol or env sequencing (6). The new assay differs from the original in two respects: first, RNA is extracted with the Magna Pure automated method (Magna Pure Large Volume; Roche Diagnostic, Meylan, France), and second, a second reverse primer is added (L140, 5'-TCCAACAGGCTCTCTGCTAATCC-3'). Thus, the 20-µl reaction mixes consisted of 7.5 µl of Light Cycler RNA master hybridization mix, 3.25 mM Mn(OAc)2, 0.5 µl of primers F3 and R1 and probe (20 µM), 0.5 µl of primer L140 at 10 µM, and 6 µl of RNA extract. The PCR conditions were as follows: (i) reverse transcription, 61°C for 20 min; (ii) denaturation, 95°C for 2 min; (iii) 45 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 20 s, and elongation at 65°C for 50 s; and (iv) cooling to 40°C for 30 s.

The specificity of the assay was determined by testing plasma samples from 25 HIV-seronegative blood donors and 25 HIV-1-infected patients. The efficiency and sensitivity of real-time reverse transcription-PCR for HIV-2 RNA quantification were determined by testing the HIV-2 RNA standard. No subtype B reference isolates being available, a stock of HIV-2 subtype A strain NIHZ, counted by electron microscopy, was used as a standard (Advanced Biotechnology Incorporated, Maryland). The NIHZ stock solution, corresponding to a subtype A strain, was diluted in HIV-negative plasma to obtain 500,000, 50,000, 5,000, 500, 250, and 100 copies/ml. It was then extracted and treated as described above. To assess the reproducibility of the assay, the standard was diluted in HIV-negative plasma and tested in eight replicates to determine within-run variability and in 10 separate runs to determine between-run variability.

Using automated extraction, the sensitivity of the assay, based on repeated testing (10 replicates each) of the standard concentrations, was 100% at 100 copies/ml. For a theoretical virus concentration of 100 copies/ml, we obtained a mean value of 124 ± 78 copies/ml with a within-run coefficient of variation of 54%. The quantification cutoff for the assay was thus set at 100 copies/ml. None of the HIV-negative or HIV-1-positive samples gave a positive result.

The Light Cycler system offers good reproducibility for HIV-2 RNA quantification. For a theoretical virus concentration of 5.69 log10 copies/ml, we obtained a mean value of 5.65 log10 copies/ml, with a within-run coefficient of variation of 1%. A mean copy number of 5.94 log10 copies/ml was obtained in repeated assays, with a between-run coefficient of variation of 0.44%. At the lowest concentration of 2.39 log10 copies/ml, we found mean values of 2.73 log10 copies/ml and 2.36 log10 copies/ml in within- and between-run assays, with coefficients of variation of 12% and 1.67%, respectively.

Among the 57 HIV-2 plasma samples tested, 28 samples, in which plasma RNA was below 100 copies with the old version, were also undetectable with the new assay (subtype A, n = 7; subtype B, n = 21). The results are summarized in Table 1. The viral load values given by the former and new versions of our assay for subtypes A and H were not different (medians were 3,790 [original version] and 3,555 [new version] copies/ml and 3,670 [original version] versus 8,030 [new version] copies/ml, P = 0.89, for subtypes A and H, respectively). The new version of the test performed significantly better than the original version with regard to the detection of HIV-2 subtype B. Indeed, among the 36 patients infected by B subtype, 14 specimens which gave negative results in the original version were positive with the new version (medians were <100 and 3,475 copies/ml).


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  		TABLE 1. Comparative results of the former assay and the optimized version for HIV-2 viral load for different subtypes

This study shows that the automation of RNA extraction by the Magna Pure system leads to increased sensitivity. Moreover, the addition of a new primer does not change the quantification of subtype A or H viral load but clearly improves the quantification of subtype B. Several groups have previously reported the failure of viral load monitoring in patients infected by divergent HIV-1 subtypes (1, 5, 9), and we report here a similar impact of HIV-2 genetic diversity on viral load assay results. Based on these results, it is likely that further refinements of HIV-2 viral load assays will be necessary to encompass the diversity of HIV-2 subtypes.


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ACKNOWLEDGMENTS
 
This study was financially supported by the Agence Nationale de Recherche sur le SIDA.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Virologie, Hôpital Bichat Claude Bernard, 46 rue Henri Huchard, 75018 Paris, France. Phone: 33 1 40 25 61 51. Fax: 33 1 40 25 67 69. E-mail: florence.damond{at}bch.ap-hop-paris.fr. Back


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REFERENCES
 
    1
  1. Alaeus, A., K. Lidman, A. Sonnerborg, and J. Albert. 1997. Subtype-specific problems with quantification of plasma HIV-1 RNA. AIDS 11:859-865.[CrossRef][Medline]
    2. 2
  2. Ariyoshi, K., S. Jaffar, A. S. Alabi, N. Berry, M. S. van der Loeff, S. Sabally, P. T. N'Gom, T. Corrah, R. Tedder, and H. Whittle. 2000. Plasma RNA viral load predicts the rate of CD4 T cell decline and death in HIV-2-infected patients in West Africa. AIDS 14:339-344.[CrossRef][Medline]
    3. 3
  3. Berry, N., K. Ariyoshi, S. Jaffar, S. Sabally, T. Corrah, R. Tedder, and H. Whittle. 1998. Low peripheral blood viral HIV-2 RNA in individuals with high CD4 percentage differentiates HIV-2 from HIV-1 infection. J. Hum. Virol. 1:457-468.[Medline]
    4. 4
  4. Chen, Z., A. Luckay, D. L. Sodora, P. Telfer, P. Reed, A. Gettie, J. M. Kanu, R. F. Sadek, J. Yee, D. D. Ho, L. Zhang, and P. A. Marx. 1997. Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys. J. Virol. 71:3953-3960.[Abstract]
    5. 5
  5. Damond, F., C. Apetrei, D. Descamps, F. Brun-Vezinet, and F. Simon. 1999. HIV-1 subtypes and plasma RNA quantification. AIDS 13:286-288.[Medline]
    6. 6
  6. Damond, F., C. Apetrei, D. L. Robertson, S. Souquiere, A. Lepretre, S. Matheron, J. C. Plantier, F. Brun-Vezinet, and F. Simon. 2001. Variability of human immunodeficiency virus type 2 (hiv-2) infecting patients living in France. Virology 280:19-30.[CrossRef][Medline]
    7. 7
  7. Damond, F., M. Gueudin, S. Pueyo, I. Farfara, D. L. Robertson, D. Descamps, G. Chene, S. Matheron, P. Campa, F. Brun-Vezinet, and F. Simon. 2002. Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections. J. Clin. Microbiol. 40:3654-3659.[Abstract/Free Full Text]
    8. 8
  8. Damond, F., M. Worobey, P. Campa, I. Farfara, G. Collin, S. Matheron, F. Brun-Vezinet, D. L. Robertson, and F. Simon. 2004. Identification of a highly divergent HIV-2 and proposal for a change in HIV-2 classification. AIDS Res. Hum. Retrovir. 20:666-672.[CrossRef][Medline]
    9. 9
  9. Debyser, Z., E. Van Wijngaerden, K. Van Laethem, K. Beuselinck, M. Reynders, E. De Clercq, J. Desmyter, and A. M. Vandamme. 1998. Failure to quantify viral load with two of the three commercial methods in a pregnant woman harboring an HIV type 1 subtype G strain. AIDS Res. Hum. Retrovir. 14:453-459.[Medline]
    10. 10
  10. Gao, F., L. Yue, D. L. Robertson, S. C. Hill, H. Hui, R. J. Biggar, A. E. Neequaye, T. M. Whelan, D. D. Ho, G. M. Shaw, et al. 1994. Genetic diversity of human immunodeficiency virus type 2: evidence for distinct sequence subtypes with differences in virus biology. J. Virol. 68:7433-7447.[Abstract/Free Full Text]
    11. 11
  11. Gottlieb, G. S., P. S. Sow, S. E. Hawes, I. Ndoye, M. Redman, A. M. Coll-Seck, M. A. Faye-Niang, A. Diop, J. M. Kuypers, C. W. Critchlow, R. Respess, J. I. Mullins, and N. B. Kiviat. 2002. Equal plasma viral loads predict a similar rate of CD4+ T cell decline in human immunodeficiency virus (HIV) type 1- and HIV-2-infected individuals from Senegal, West Africa. J. Infect. Dis. 185:905-914.[CrossRef][Medline]
    12. 12
  12. Kanki, P. J., K. U. Travers, S. MBoup, C. C. Hsieh, R. G. Marlink, N. A. Gueye, T. Siby, I. Thior, M. Hernandez-Avila, J. L. Sankale, et al. 1994. Slower heterosexual spread of HIV-2 than HIV-1. Lancet 343:943-946.[CrossRef][Medline]
    13. 13
  13. Marlink, R., P. Kanki, I. Thior, K. Travers, G. Eisen, T. Siby, I. Traore, C. C. Hsieh, M. C. Dia, E. H. Gueye, et al. 1994. Reduced rate of disease development after HIV-2 infection as compared to HIV-1. Science 265:1587-1590.
    14. 14
  14. Matheron, S., S. Pueyo, F. Damond, F. Simon, A. Lepretre, P. Campa, R. Salamon, G. Chene, and F. Brun-Vezinet. 2003. Factors associated with clinical progression in HIV-2 infected patients: the French ANRS cohort. AIDS 17:2593-2601.[CrossRef][Medline]
    15. 15
  15. Yamaguchi, J., S. G. Devare, and C. A. Brennan. 2000. Identification of a new HIV-2 subtype based on phylogenetic analysis of full-length genomic sequence. AIDS Res. Hum. Retrovir. 16:925-930.[CrossRef][Medline]

Journal of Clinical Microbiology, August 2005, p. 4234-4236, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4234-4236.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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  • Damond, F., Benard, A., Ruelle, J., Alabi, A., Kupfer, B., Gomes, P., Rodes, B., Albert, J., Boni, J., Garson, J., Ferns, B., Matheron, S., Chene, G., Brun-Vezinet, F., for the ACHIEV2E (a Collaboration on HIV-2 Infecti, (2008). Quality Control Assessment of Human Immunodeficiency Virus Type 2 (HIV-2) Viral Load Quantification Assays: Results from an International Collaboration on HIV-2 Infection in 2006. J. Clin. Microbiol. 46: 2088-2091 [Abstract] [Full Text]  

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