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J Clin Microbiol. Jun 2008; 46(6): 2088–2091.
Published online Apr 23, 2008. doi:  10.1128/JCM.00126-08
PMCID: PMC2446851

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[down-pointing small open triangle]

Abstract

Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHIEV2E (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each at a different theoretical viral load. Quantification varied between laboratories, and international standardization of quantification assays is strongly needed.

The human immunodeficiency virus type 2 (HIV-2) epidemic remains essentially confined to West Africa (4). In Europe, most of the patients live in France, Portugal, and Spain, although patients are also found in The Netherlands, Belgium, Luxemburg, Germany, Switzerland, Italy, the United Kingdom, and Sweden (5, 10-13). Many questions remain unanswered concerning the evolution of virological, immunological, and clinical parameters under treatment. Answering these questions requires the largest possible number of observations and standardization of outcome measurements such as that of the HIV-2 load in plasma. There is currently no available commercial assay for the quantification of plasma HIV-2 RNA. In this study, we evaluated the validity of assays used for the quantification of HIV-2 RNA among nine centers in the ACHIEV2E (A Collaboration on HIV-2 Infection) study group, in Belgium, France, Gambia, Germany, Portugal, Spain, Sweden, Switzerland, and the United Kingdom, in order to identify appropriate actions to improve the quality of these assays. Each of the nine participating laboratories performed a different HIV-2 RNA quantification assay (2, 3, 7-9). Laboratories used mainly different “in-house” real-time PCR assays with primers and probes located in various regions of the HIV-2 genome, and they employed different HIV-2 standards (Table (Table11).

TABLE 1.
Characteristics of the different quantification assays assessed by the ACHIEV2E Collaboration in 2006

The sample panel was prepared in a single virology laboratory (Bichat-Claude Bernard Hospital, Paris, France) by performing serial dilution of the NIHz HIV-2 subtype A supernatant, quantified by electron microscopy (ABI Technologies), in HIV-negative human plasma to obtain 10 1-ml aliquots each with final HIV-2 RNA concentrations of 1.7, 2.3, and 3.0 log10 copies/ml.

The accuracy and reproducibility of assays were estimated for the three theoretical concentrations. A quantification assay was defined as accurate if at least 9 of the 10 measurements fell within a clinically acceptable interval based on the NIHz theoretical concentrations. The interval was defined as one-third of the theoretical viral load to three times the theoretical viral load for theoretical viral loads of 3.0 and 2.3 log10 copies/ml and as 0 to three times the theoretical viral load for a theoretical viral load of 1.7 log10 copies/ml.

Reproducibility of assays was evaluated by using the intralaboratory coherence coefficient (ILCC) and the coefficient of variation (CV) at each of the three theoretical concentrations (1). A quantification heterogeneity was defined as an ILCC greater than the theoretical value L, extracted from a reference table for p laboratories and n repetitions (1).

Each of the participating laboratories quantified 35 coded and randomized aliquots. All laboratories reported an undetectable viral load for the five HIV-negative aliquots.

At a theoretical level of 3.0 log10 copies/ml, median quantifications varied from 2.9 log10 copies/ml in laboratory 6 to 4.2 log10 copies/ml in laboratory 8 (Fig. (Fig.1A).1A). Five laboratories reported accurate measurements (i.e., 9 of the 10 quantifications fell in the interval from 2.52 to 3.48 log10 copies/ml). The four other assays overquantified viral loads (laboratories 1, 3, 7, and 8) (Fig. (Fig.1A).1A). Seven laboratories reported reproducible measurements (CV = 3 to 6%; ILCC = 0.5 to 0.9; L = 1.35) (laboratories 1, 2, 3, 6, 7, 8, and 9). Laboratories 2, 6, and 9 reported both accurate and reproducible measurements. At a theoretical level of 2.3 log10 copies/ml, the accuracy and reproducibility of the tests at laboratories 4 and 5 could not be evaluated since 10 and 9 negative results were reported, respectively. Median quantifications varied from 2.4 log10 copies/ml in laboratory 9 to 3.4 log10 copies/ml in laboratory 8 (Fig. (Fig.1B).1B). Three laboratories reported accurate measurements (i.e., 9 of the 10 quantifications fell in the interval from 1.82 to 2.78 log10 copies/ml), and four laboratories overquantified viral loads (laboratories 1, 3, 7, and 8) (Fig. (Fig.1B).1B). Six laboratories reported reproducible measurements (CV = 5 to 8%; ILCC = 0.5 to 1.1; L = 1.35). Only two laboratories, 2 and 9, reported both accurate and reproducible measurements.

FIG. 1.
Accuracy of quantification assays evaluated by the ACHIEV2E Collaboration in 2006. Quantification results are reported for each participating laboratory. The accuracy interval is represented by the white area for each of the three theoretical viral loads ...

At a theoretical level of 1.7 log10 copies/ml, the accuracy and reproducibility of the assays used by laboratories 4, 5, 6, and 7 could not be evaluated since 9, 10, 7, and 9 negative results were reported, respectively. The median quantifications varied from 1.4 log10 copies/ml in laboratory 2 to 2.9 log10 copies/ml in laboratory 8 (Fig. (Fig.1C).1C). Two laboratories, 2 and 9, reported accurate measurements (i.e., 9 of the 10 quantifications were below 2.18 log10 copies/ml), and three laboratories overquantified viral loads (laboratories 1, 3, and 8) (Fig. (Fig.1C).1C). These three laboratories reported reproducible measurements (CV = 5 to 11%; ILCC = 0.4 to 0.8; L = 1.35). At this level, no laboratories reported both accurate and reproducible measurements.

Our study is the first international evaluation of the validity of HIV-2 RNA quantification assays. We found a fair homogeneity of results of assays evaluated at a theoretical level of 3.0 log10 copies/ml and a decreasing accuracy and reproducibility of assays with decreasing viral loads, as previously shown in a quality control study of HIV-1 RNA quantification assays (6). Overall, four laboratories systematically overquantified viral loads, no. 1, 3, 7, and 8. Two laboratories, 4 and 5, reported 18 negative results out of 20 quantifications at theoretical viral loads of both 2.3 and 1.7 log10 copies/ml. The thresholds of these two assays had been evaluated at 2.7 and 2.3 log10 copies/ml, respectively. The cutoff values of the methods used by laboratories 5 and 7 may also explain their results for the 1.7-log10-copy/ml aliquots. One laboratory (no. 6) reported both accurate and reproducible results at a theoretical viral load of 3.0 log10 copies/ml but heterogeneous quantifications at 2.3 log10 copies/ml and negative results at 1.7 log10 copies/ml. Two laboratories, 2 and 9, reported both accurate and reproducible results at theoretical viral loads of 3.0 and 2.3 log10 copies/ml and accurate but relatively poorly reproducible quantifications at 1.7 log10 copies/ml. The aliquot were made from the NIHz HIV-2 supernatant, which is the standard used for the calibration of the laboratory 2 assay. That may have improved the results reported by this laboratory and decreased those observed in other laboratories such as no. 8, which has not yet been evaluated with calibrated specimens. However, laboratory 9, which did not use NIHz as a standard, reported results very similar to those obtained by laboratory 2.

This study provides an overview of the performance of different HIV-2 RNA quantification assays. If the HIV-2 supernatant counted by electron microscopy is accepted as a standard, only two laboratories reported accurate and reproducible measurements at both 3 and 2.3 log10 copies/ml. This heterogeneity may yield difficulties in the comparison of results between the different cohorts of the ACHIEV2E Collaboration. The ACHIEV2E network is working toward the standardization of quantification assays to improve the interpretation of results for case management and collaborative clinical trials.

Acknowledgments

This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS).

The members of the ACHIEV2E study group in Belgium are Patrick Goubau and Jean Ruelle (AIDS Reference Laboratory, Université Catholique de Louvain). Those in France are Françoise Brun-Vezinet, Pauline Campa, Florence Damond, Diane Descamps, Sophie Matheron (Bichat—Claude Bernard Hospital, Paris), François Simon (Saint-Louis Hospital, Paris), Antoine Benard, Geneviève Chene, Audrey Taieb (INSERM U897, Bordeaux2 University, Bordeaux), and Brigitte Autran (Pitié-Salpétrière Hospital, Paris). Those in Gambia are Abraham Alabi, Matt Cotten, Assan Jaye, Kevin Peterson, and Sarah Rowland-Jones (Medical Research Council Laboratories). Those in Germany are Bernd Kupfer (Institute of Virology, Bonn), Jürgen Rockstroh, and Carolynne Schwarze-Zander (Department of Internal Medicine I, University of Bonn, Bonn). Those in The Netherlands are Frank De Wolf, Ard Van Sighem (National AIDS Therapy Evaluation Center, AMC, Amsterdam), Peter Reiss, Maarten Schim Van Der Loeff (AMC-CPCD, Amsterdam; MRC, Gambia), and Martin Schutten (Department of Virology, Erasmus MC, Rotterdam). Those in Portugal are Ricardo Camacho, Perpetua Gomes, Kamal Mansinho (Egas Moniz Hospital, Lisbon), Francisco Antunes, Luis Franca, and Emilia Valadas (Clinica Universitaria de Doenças Infecciosas, Lisbon). Those in Spain are Berta Rodes, Carlos Toro, and Vicente Soriano (Department of Infectious Diseases, Hospital Carlos III, Madrid). Those in Sweden are Jan Albert (Department of Virology, Swedish Institute for Infectious Disease Control, Solna), Katarina Gyllensten, Anders Sonnerborg (Divisions of Clinical Virology and Infectious Diseases, Huddinge University Hospital, Karolinska Institute, Stockholm), Aylin Yilmaz, and Magnus Gisslén (Department of Infectious Diseases, Sahlgrenska University Hospital, Göteborg). Those in Switzerland are Jürg Böni, Alexandra Calmy, and Martin Rickenbach (Swiss HIV Cohort Study). Those in the United Kingdom are Deenan Pillay, Bridget Ferns, Jeremy Garson (Centre for Virology, Royal Free & University College London Medical School, London), Jennifer Tosswill (Centre for Infections, Health Protection Agency, London), Jane Anderson (Centre for the Study of Sexual Health and HIV, Homerton University Hospital, London), and David Chadwick (James Cook University Hospital, Middlesbrough).

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 April 2008.

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