Journal of Clinical Virology 58S (2013) e70–e75
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Field evaluation of the Abbott ARCHITECT HIV Ag/Ab Combo immunoassay Anfumbom Kfutwah a,∗ , Véronique Lemée b , Hélène Valérie Ngono a , Fabienne De Oliveira b , Richard Njouom a , Jean-Christophe Plantier b,∗∗ a Service de Virologie, Centre Pasteur du Cameroun, Membre du Réseau International des Instituts Pasteur, Yaounde’, Cameroun. BP 1274, Yaounde, Cameroon b Laboratoire Associé au Centre National de Référence du VIH, Hôpital Charles Nicolle, CHU de Rouen, 76031 Rouen, France
a r t i c l e
i n f o
Article history: Received 14 June 2013 Received in revised form 9 August 2013 Accepted 17 August 2013 Keywords: Africa HIV immunoassay Genetic diversity Field evaluation
a b s t r a c t Background: Fourth generation assays for HIV diagnosis are progressively being introduced into routine services, due to their improvement of diagnosis. In spite of this, HIV diagnosis remains a challenge in sub-Saharan Africa, due to false positive reactivity. There is a continuous need for field evaluations and routine validations of fourth generation HIV tests in African populations. Objectives: Evaluate the performances of the ARCHITECT HIV Ag/Ab kit (Abbott) in a population living in an African setting-Cameroon compared to a population living in a European setting-France. Study design: 645 HIV samples from both France and Cameroon were evaluated. The positive panel (378 samples) included a diverse series of HIV-1 variants (groups M, N, O, and P) as well as HIV-2 samples. Results were compared to original diagnosis done with other 4th generation assays (AxSYM HIV Ag/Ab (Abbott) and Vidas HIV DUO QUICK) (bioMérieux). Results: Sensitivity of the ARCHITECT was 100% in both sites. It diagnosed all variants of the panel with different reactivity profiles following strain diversity. A wider range of reactivity was observed for group O. Specificity was slightly lower (97.6%) in Cameroon than in France (98.6%), probably due to a higher rate of false positive reactivity. ARCHITECT HIV Ag/Ab assay had high performances in clinical sensitivity and specificity and is adapted to the wide genetic diversity of viruses circulating in West Central Africa. Conclusion: Our results further highlight the need to evaluate HIV diagnostic tests before introduction into routine diagnostic services both in the North and in the South. © 2013 Elsevier B.V. All rights reserved.
1. Background “Fourth-generation” HIV immunoassays, which reduce the seroconversion period by 4–8 days [1,2], allows for the detection of early and established infections [1]. More than 10 different fourthgeneration assays have been licensed in Europe [3,4] and HIV automated fourth-generation tests are increasingly being used in diagnostic laboratories [5–7]. Several assay evaluations have been carried out in the Northern hemisphere [5,6,8], mainly on samples with Caucasian origin [6,9]. In sub-Saharan Africa (particularly in central Africa), serological diagnosis remains challenging due to
Abbreviations: HIV, human immunodeficiency virus; RT, reverse transcriptase; PCR, polymerase chain reaction. ∗ Corresponding author. Tel.: +237 22 23 10 15; fax: +237 22 23 15 64. ∗∗ Corresponding author at: Laboratoire de Virologie, Institut de Biologie Clinique, Hôpital, Charles Nicolle, CHU de Rouen, 1 rue de Germont, 76031 Rouen, France. Tel.: +33 2 32 88 14 62; fax: +33 2 32 88 04 30. E-mail addresses: [email protected], [email protected] (A. Kfutwah), [email protected] (J.-C. Plantier). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.08.015
the large genetic diversity of circulating HIV strains, and also false results notably cross reactivity observed with tuberculosis [10] and leprosy patients [11]. Other studies have suggested agents, such as schistosomiasis, malaria and human T-cell lymphotropic virus as responsible for this cross reactivity, but these effects have not been demonstrated consistently [12–14]. 2. Objectives As few fourth generation HIV diagnostic test evaluations have been carried out in sub-Saharan Africa, we evaluated the sensitivity and specificity of the ARCHITECT HIV Ag/Ab Combo kit (Abbott Laboratories, GmBH, Delkenheim, Germany) in a population living in an African setting-Cameroon compared to a population living in a European setting-France. 3. Study design 645 samples from Cameroon and France were evaluated in this study. 250 plasma samples from 250 patients were collected and
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Table 1 Amino acid sequences of the peptides used for HIV “2 peptides” and “10 peptides” serotyping formats. Peptides used
Peptide sequences
Gp41 M Gp41 N Gp41 O Gp41 Sm V3 M V3 N V3 O (clade A)a V3 O (consensus) V3 P V3 Sm
RVLAVERYLKDQQLLGIWGCSGKLICTTAV LAIERYLRDQQILSLWGCSGKTIC LALETLIQNQQLLNLWGCKGKLIC TAIEKYLKDQAQLNSWGCAFRQVC NNTRKSVHIGPGQAFYATGDIIGDIRQAHC NNTGGQVQIGPAMTFYNIEKIVGDIRQA IDIQEMRIGPMAWYSMGIGGTAGNSSRAA NLTVQEIKIGPMAWYSMGLAAGNGNSRAYC NNTRGQIQVGPLTIYNSENIIGNTRKAF NKTVLPVTIMSGLVFHSQPINERPKQA
Two peptides
X X
Ten peptides X X X X X X X X X X
a V3 O (clade A) represent the majority clade in circulation in Cameroon while V3 O (consensus) covers the entire antigenic diversity of HIV group O. X indicate peptides used “10 peptides” format and X indicate peptides used for the “2 peptides format”.
characterized in the HIV reference laboratory of Centre Pasteur in Cameroon (CPC). 395 fresh or frozen serum and plasma samples from 377 patients were collected and characterized in the laboratory associated to the National HIV Reference Centre of the Rouen University Hospital (CHU) in France. HIV samples routinely tested by 4th generation tests; AxSYM HIV Ag/Ab Combo (Abbott Laboratories) in CPC, and Vidas HIV DUO QUICK (bioMérieux, Marcy l’Etoile, France) in Rouen were re-tested with the ARCHITECT HIV Ag/Ab Combo test kit. Architect HIV Ag/Ab Combo is a chemiluminescent magnetic microparticle-based immunoassay used to determine the presence of HIV-1 p24 antigen and antibody to HIV-1 groups M and O, and HIV-2 by an automated, random access instrument [8]. Sample/Cutoff ratios (S/CO) of >1 were considered reactive and indicated the presence of anti-HIV immunoglobulin and/or p24 antigen; ratios equal to or greater than 0.90 and less than 1.0 were in the gray zone; and ratios of 0.25 indicated the presence of anti-HIV immunoglobulin and/or p24 antigen. For easy representation and comparison of the S/CO with other kits, indexes were calculated relative to the threshold of 1. Samples with discordant results between the “routine” tests and ARCHITECT were analyzed with “local” algorithms in the different laboratories: In Cameroon, samples were repeated in duplicates (ARCHITECT) and further characterized with a 3rd generation test (detection of antibodies only), Genscreen HIV-1/2 version 2 (BIO-RAD, Marnes La Coquette, France) and confirmation assays of western blot (WB) New LAV BLOT 1 and II (BIO-RAD) following the manufacturer’s instructions. Nucleic acid testing (NAT) was performed: briefly, viral RNA was extracted from 1 mL of plasma using the Qiagen viral RNA mini kit (Qiagen, Inc., Chatsworth, CA) as recommended by the manufacturer. The RT (codons 1–255) and Protease genes (codons 1–99) of HIV-1 group M were amplified by Reverse transcriptase PCR using the primers MJ3/MJ4 and 51Prot1/31Prot1, respectively, followed by nested PCR with primers A35/NE1 for RT and 51 Prot 2/31 Prot 2 for protease [15,16]. Visualization of amplified products was done in a 1.5% agarose gel. Discordant samples were tested with an in-house serotyping test as previously described [17], with slight modifications. This inhouse peptide linked immunosorbent assays (PLISA) use synthetic peptides mapping the V3 and gp41/36 regions of HIV (Table 1) to differentiate into the different serotypes (HIV-1: M, O, N, P,
and HIV-2). A first test, “two peptides”, explored the V3 region of HIV-1 groups M (HIV-M) and O (HIV-O). Negative samples in the “two peptides” assay were re-tested in a second PLISA “ten peptides” using peptides mapping the gp41/36 region of HIV-1 groups M, O, N, HIV-2/SIVsm on one hand and V3 peptides of M, O (clade A and consensus), N, P, HIV-2/SIVsm on the other (Table 1). In France, samples were analyzed with (i) 3rd generation kit AxSYM HIV1/2 gO (Abbott), (ii) WB (BIO-RAD), and (iii) Vidas HIV P24 II (bioMérieux) for the determination of the p24 antigen of HIV-1. These tests were performed according to the manufacturer’s instructions, in two consecutive samples collected 15 days apart. Strain diversity was explored on all the positive samples, in Cameroon, using the serotyping assay described above and in France, using the test developed by Simon et al. [18] 4. Results The mean age of the 250 persons tested in Cameroon was 32.4 years (2–68 years) comprising 151 females, 91 males, and 8 children (2–3 years) whose genders were inaccessible. 141/250 (56.4%) had been characterized as HIV positive with AxSYM (Fig. 1a). Of these 141 samples, 126 (89.3%) were also reactive in ARCHITECT with a mean S/CO of 545.34 (range: 1.3–1097.9), and corresponding mean S/CO in AxSYM of 28.7 (range: 1.16–61.88). Fifteen (10.6%) samples were reactive for AxSYM (mean 2.38; range: 1.12–7.63) and non-reactive for ARCHITECT. These 15 discordant samples further tested by Genscreen, WB I and II, NAT and serotyping were nonreactive. On the other hand, 106 of the 109 (97.3%) samples that were non-reactive with AxSYM were also non-reactive with ARCHITECT (Fig. 1a). The remaining 3 (2.7%) ARCHITECT reactive had the following ratios: 10.74, 29.59, and 33.56. These 3 samples were non reactive in Genscreen, WB 1 and II, NAT and serotyping assays. Finally, of the 250 samples tested for both AxSYM and ARCHITECT, 232 (92.8%) were concordant for both assays while 18 (7.2%) were discordant. AxSYM and ARCHITECT misdiagnosed 15 and 3 HIV negative samples respectively. The sensitivity and specificity were 100% (95% CI: 96.3–100%) and 87.9% (95% CI: 80.5–92.8%), respectively for AxSYM, and 100% (95% CI: 96.3–100%) and 97.6% (95% CI: 92.6–99.3), respectively, for ARCHITECT. The mean age of the 377 persons tested in France was 40.7 years (1–86 years) comprising 148 females, 226 males, and 3 persons whose genders were inaccessible. 268/395 samples (67.8%) had been characterized as HIV positive in Vidas (relative index >1) (Fig. 1b). Among these 268 samples, 252 (94%) were also reactive in ARCHITECT with a mean S/CO of 391.79 (range 2.15–852.16), and corresponding mean S/CO of 84.83 (range 4.24–128) in Vidas. Sixteen samples (6%) were reactive for Vidas (mean ratio 2.71; range 1.12–14.72) and non-reactive for ARCHITECT. These discordant samples were further tested with the algorithm defined in CHU Rouen, repeated 2 weeks later on new samples, and all were negative indicating false positive reactions.
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a
250 Samples 141 Reacve AxSYM
126 Reacve Architect
109 Non-Reacve AxSYM 15 Non-Reacve Architect
106 Non-Reacve Architect
3 Reacve Architect
negave with all complementary tools
negave with all complementary tools
b 395 Samples 268 Reacve Vidas 252 Reacve Architect
127 Non reacve Vidas 16 Non reacve Architect
125 Non reacve Architect
2 Reacve Architect
negave with all complementary tools
negave with all complementary tools* for one not enough material
Fig. 1. (a) Comparison of sample reactivity between AxSYM and ARCHITECT in Cameroon. (b) Comparison of sample reactivity between Vidas and ARCHITECT in France.
Of the 127 samples negative with Vidas, 125 were also non-reactive with ARCHITECT (98.4%) (Fig. 1b). The two (1.6%) ARCHITECT reactive samples had the following ratios: 3.22 and 2.44. For one, complementary test (AxSYM HIV1/2 gO) was negative and also negative for the sample collected 2 weeks later. No further tests were carried out for the second due to insufficient sample. Finally, of the 395 samples tested for both Vidas and ARCHITECT, 377 (95.4%) were concordant while 18 (4.5%) were discordant. The sensitivity and specificity were 100% (95% CI: 98.1–100%) and
88.8% (95% CI: 82.2–93.2%) respectively for Vidas, and 100% (95% CI: 98.1–100%) and 98.6% (95% CI: 94.5–99.7%) respectively, for ARCHITECT. In Cameroon, among the 126 samples positive for both AxSYM and ARCHITECT, 110 (87.3%) were serotyped as HIV-M, and 11 (8.7%) as HIV-O (Table 2). Three other samples presented dual reactivity in groups M and O. Finally two samples, with low S/CO values of 6.66 and 3.65 for AxSYM, and 1.3 and 5.54 for ARCHITECT, were negative for serotyping. Because of sample exhaustion no further molecular tests were carried out. In France, the 252 positive
Table 2 Cutoff ratios of the different samples from Cameroon and France, in the three tests.
Group M, Cameroon (n = 110) Group M, France (n = 216) Group O, Cameroon (n = 11) Group O, France (n = 23) Group M + O, Cameroon (n = 3) Group N, France (n = 1) Group P, France (n = 1) HIV 2, France (n = 9) HIV 1 + 2, France (n = 2)
AxSYM HIV Ag/Ab combo (Abbott)
ARCHITECT HIV Ag/Ab Combo (Abbott)
Vidas HIV DUO Quick (bioMérieux)
Mean ratio
Maximum ratio
Minimum ratio
Mean ratio
Maximum ratio
Minimum ratio
Mean ratio
Maximum ratio
Minimum ratio
28.98
61.88
2.98
490.84
1097.92
5.94
/
/
/
/
/
/
438.60
852.16
8.65
85.71
128
6.04
14.44
30.75
5.74
107.92
382.6
7.73
/
/
/
/
/
/
40.78
224.97
2.15
76.96
92.68
4.24
19.43
27.42
18.38
521.43
808.24
14.89
/
/
/
/
/
/
NA (21.42)
NA
NA
NA (16.92)
NA
NA
/
/
/
NA
NA
NA (52.2)
NA
NA
/ /
/ /
/ /
NA (537.95) 118.26 NA (712.83 and 719.36)
132.01 NA
94.87 NA
92.53 NA (92.64 and 99.68)
101.36 NA
85.36 NA
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Fig. 2. Logarithmic representation of HIV-1 groups M, O and M/O reactivities ratio for samples from Cameroon and France.
samples for both Vidas and ARCHITECT provided a representative diversity of strains circulating in the country defined by the following serotypes (Table 2): HIV-M (n = 216; 214 patients), HIV-O (n = 23; 22 patients), HIV-N (n = 1), HIV-P (n = 1), HIV-2 (n = 9), and two samples HIV-1 + HIV-2 dual reactive (n = 2). The samples positive with ARCHITECT presented different values according to the serogroups (Table 2, Fig. 2). HIV-M values were concentrated toward the high range of 100–1000 (Fig. 2), both in Cameroon (mean ratio of 490.84) and France (mean ratio of 438.60) (Table 2). The HIV-O had lower mean ratios (107.92 and 40.78 for Cameroon and France respectively) (Table 2), and are more widespread with maximum ratio not above 400 (Fig. 2). These results indicate a clear difference of reactivity between these two groups. Similar observations were made for the lower HIV-2 mean ratio of 118.26 compared to those of HIV-M. Whenever there is dual reactivity, HIV-M + O or HIV-1 + 2, the sample reactivity is comparable to HIV-M (Table 2). The lone HIV-P sample (derived from SIV infecting gorilla [19]), reacted as HIV-M despite its genetic links with group O. The weak reactivity of HIV-N, could be associated to the acute nature of the sample (seroconversion phase) rather than its antigenic divergence [20]. Of note, AxSYM values are generally lower than those with ARCHITECT, with a lower gradient of reactivity, but similar reactivity between HIV-M and O (Table 2, Fig. 2). The reactivities obtained with the Vidas are more concentrated irrespective of genetic diversity. These results indicate that ARCHITECT reactivity allows the “suspicion” of the serotype/serogroup of the infecting strains, better than AxSYM and Vidas that are more homogeneous. 5. Discussion Several studies have evaluated ARCHITECT in different geographical locations with different HIV prevalence [4,7,21–25]. Here, we conducted a large field evaluation on 645 samples representing
various clinical situations from populations living in sub-Saharan Africa and Europe, permitting a direct comparison of the results. Moreover, this panel is representative to a great extent of the HIV genetic diversity, including distinct variants that are circulating in Europe and in Central Africa, with the rare variants of groups N and P. The results show that the ARCHITECT Combo assay correctly detected all HIV positive samples, whatever the type of population and the clinical stage. This high sensitivity concur previous observations [7,22] highlighting its usefulness in determining established and recent infections [23,25]. Furthermore the presence of antigens representative of HIV-1 group M, O and HIV-2 allows for the detection of a large specter of HIV antibodies even those directed toward the rare groups of HIV-1 group N and P, thereby limiting false negative results in environments of high genetic variability. Our findings therefore complement the evaluations that have been done by others [7,8,22,26,27]. S/CO ratios observed with ARCHITECT are different following the serotype/serogroup, with generally higher ratios compared to AxSYM and Vidas. The absence of a complete correlation between ratios and HIV variants do not allow for the identification of the serotype/serogroup based on the S/CO values only, nevertheless these values could be very informative. An important characteristic of group O samples was their comparatively low and heterogeneous ratios (2.15–382.6), compared to group M ratios; this was also observed for HIV-2 but with a lower ratio (around 100) and a lower widespread distribution. These lower values could highlight the divergence at the antigenic level between group O, HIV-2 and group M. The heterogeneous values observed for HIV-O could be explained by the genetic diversity found among this group particularly at the immunodominant epitope region of the envelope protein, which serves as a target for detection [28,29]. As a consequence of the widespread S/CO ratios observed with diverse strains, the classical S/CO values observed for false positive
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samples could be misleading. In a previous study, in a low risk population [30], the S/CO of false-positive results with ARCHITECT was 2.94 (range: 1.00–34.59). It was also observed that false positive reactivity was higher when this ratio of ARCHITECT was in the low range of 1–5 [23]. Here, we observed a higher mean S/CO of 15.9 (2.44–33.56) for false positive samples from both Cameroon and France. These values are generally lower than the values obtained for HIV-1 group M samples, but closer to values of strains such as HIV-1 group O (lowest ratio of 2.15), which could be wrongly taken for a false positive reactivity. Thus, such low values need complementary analyses to be performed particularly in regions with high HIV genetic diversity, which could definitely distinguish HIV variants from false positive reactivity. Diagnostic laboratories therefore will need to adapt to these new ranges of values and take a lot of caution when interpreting test results. Our results show an improvement in the specificities of ARCHITECT (97.6% and 98.6% for Cameroon and France respectively) compared to AxSYM (87.9%) and Vidas (88.8%). It should nevertheless be noted that these results were obtained from samples preselected by these two tests, which could introduce some bias to the analysis. However, we corroborate earlier observations where ARCHITECT was shown to have a higher specificity than AxSym [8] or other tests [4,7]. The specificities in Cameroon (97.6%) fall short of the WHO recommendations of specificities of 98% [31]. In Africa, endemic infections affect the performances of HIV diagnostic tests and particularly the specificities as a result of cross reactivity [32]. Low specificities with fourth-generation tests have been reported in Cameroon, Tanzania and Uganda [27,33] and could suggest some peculiarities involved with HIV diagnosis in these populations. A fourth generation HIV assay, Abbott Murex HIV antigen/antibody EIA, has been particularly shown in Tanzanian adolescents to have a very low specificity of 91.5% [33]. However, the specificities observed in Cameroon with ARCHITECT was not very different from that observed in France (97.6 versus 98.6%). This could be due to some samples tested in the French site, from persons of African origin living in France. Our results further highlight the fact that serological tests for HIV may perform differently in different populations. ARCHITECT, with a marked improvement in specificity compared to AxSYM and Vidas, should reduce the number of false positive samples, and thereby evade the inconveniences of such results, i.e. increased anxiety for persons with false positive results and the need for other more direct tests such as NAT and/or ELISAs/WB for confirmation, which are costly and time consuming. 6. Conclusion This study shows that the ARCHITECT HIV Ag/Ab Combo assay presents an excellent sensitivity. The evaluation on a large panel of samples from various clinical stages and representative of the high HIV genetic diversity, argues for high performances of clinical sensitivity. However, the widespread distribution of ratios for positive samples, particularly with divergent HIV strains, could cover the range of values for false positive samples. Caution is therefore needed in the interpretation of very low values, which could be in favor of an atypical reactivity corresponding to either HIV variant, an acute infection or false positive reactivity. Our study thus promotes the need to evaluate HIV diagnostic tests before introduction into routine diagnostic services, both in the North as well as in the South, where other confounding factors could be involved. Competing interests The authors declare that they have no competing interests.
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Field evaluation of the Abbott ARCHITECT HIV Ag/Ab Combo immunoassay Anfumbom Kfutwah a,∗ , Véronique Lemée b , Hélène Valérie Ngono a , Fabienne De Oliveira b , Richard Njouom a , Jean-Christophe Plantier b,∗∗ a Service de Virologie, Centre Pasteur du Cameroun, Membre du Réseau International des Instituts Pasteur, Yaounde’, Cameroun. BP 1274, Yaounde, Cameroon b Laboratoire Associé au Centre National de Référence du VIH, Hôpital Charles Nicolle, CHU de Rouen, 76031 Rouen, France
a r t i c l e
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Article history: Received 14 June 2013 Received in revised form 9 August 2013 Accepted 17 August 2013 Keywords: Africa HIV immunoassay Genetic diversity Field evaluation
a b s t r a c t Background: Fourth generation assays for HIV diagnosis are progressively being introduced into routine services, due to their improvement of diagnosis. In spite of this, HIV diagnosis remains a challenge in sub-Saharan Africa, due to false positive reactivity. There is a continuous need for field evaluations and routine validations of fourth generation HIV tests in African populations. Objectives: Evaluate the performances of the ARCHITECT HIV Ag/Ab kit (Abbott) in a population living in an African setting-Cameroon compared to a population living in a European setting-France. Study design: 645 HIV samples from both France and Cameroon were evaluated. The positive panel (378 samples) included a diverse series of HIV-1 variants (groups M, N, O, and P) as well as HIV-2 samples. Results were compared to original diagnosis done with other 4th generation assays (AxSYM HIV Ag/Ab (Abbott) and Vidas HIV DUO QUICK) (bioMérieux). Results: Sensitivity of the ARCHITECT was 100% in both sites. It diagnosed all variants of the panel with different reactivity profiles following strain diversity. A wider range of reactivity was observed for group O. Specificity was slightly lower (97.6%) in Cameroon than in France (98.6%), probably due to a higher rate of false positive reactivity. ARCHITECT HIV Ag/Ab assay had high performances in clinical sensitivity and specificity and is adapted to the wide genetic diversity of viruses circulating in West Central Africa. Conclusion: Our results further highlight the need to evaluate HIV diagnostic tests before introduction into routine diagnostic services both in the North and in the South. © 2013 Elsevier B.V. All rights reserved.
1. Background “Fourth-generation” HIV immunoassays, which reduce the seroconversion period by 4–8 days [1,2], allows for the detection of early and established infections [1]. More than 10 different fourthgeneration assays have been licensed in Europe [3,4] and HIV automated fourth-generation tests are increasingly being used in diagnostic laboratories [5–7]. Several assay evaluations have been carried out in the Northern hemisphere [5,6,8], mainly on samples with Caucasian origin [6,9]. In sub-Saharan Africa (particularly in central Africa), serological diagnosis remains challenging due to
Abbreviations: HIV, human immunodeficiency virus; RT, reverse transcriptase; PCR, polymerase chain reaction. ∗ Corresponding author. Tel.: +237 22 23 10 15; fax: +237 22 23 15 64. ∗∗ Corresponding author at: Laboratoire de Virologie, Institut de Biologie Clinique, Hôpital, Charles Nicolle, CHU de Rouen, 1 rue de Germont, 76031 Rouen, France. Tel.: +33 2 32 88 14 62; fax: +33 2 32 88 04 30. E-mail addresses: [email protected], [email protected] (A. Kfutwah), [email protected] (J.-C. Plantier). 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.08.015
the large genetic diversity of circulating HIV strains, and also false results notably cross reactivity observed with tuberculosis [10] and leprosy patients [11]. Other studies have suggested agents, such as schistosomiasis, malaria and human T-cell lymphotropic virus as responsible for this cross reactivity, but these effects have not been demonstrated consistently [12–14]. 2. Objectives As few fourth generation HIV diagnostic test evaluations have been carried out in sub-Saharan Africa, we evaluated the sensitivity and specificity of the ARCHITECT HIV Ag/Ab Combo kit (Abbott Laboratories, GmBH, Delkenheim, Germany) in a population living in an African setting-Cameroon compared to a population living in a European setting-France. 3. Study design 645 samples from Cameroon and France were evaluated in this study. 250 plasma samples from 250 patients were collected and
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Table 1 Amino acid sequences of the peptides used for HIV “2 peptides” and “10 peptides” serotyping formats. Peptides used
Peptide sequences
Gp41 M Gp41 N Gp41 O Gp41 Sm V3 M V3 N V3 O (clade A)a V3 O (consensus) V3 P V3 Sm
RVLAVERYLKDQQLLGIWGCSGKLICTTAV LAIERYLRDQQILSLWGCSGKTIC LALETLIQNQQLLNLWGCKGKLIC TAIEKYLKDQAQLNSWGCAFRQVC NNTRKSVHIGPGQAFYATGDIIGDIRQAHC NNTGGQVQIGPAMTFYNIEKIVGDIRQA IDIQEMRIGPMAWYSMGIGGTAGNSSRAA NLTVQEIKIGPMAWYSMGLAAGNGNSRAYC NNTRGQIQVGPLTIYNSENIIGNTRKAF NKTVLPVTIMSGLVFHSQPINERPKQA
Two peptides
X X
Ten peptides X X X X X X X X X X
a V3 O (clade A) represent the majority clade in circulation in Cameroon while V3 O (consensus) covers the entire antigenic diversity of HIV group O. X indicate peptides used “10 peptides” format and X indicate peptides used for the “2 peptides format”.
characterized in the HIV reference laboratory of Centre Pasteur in Cameroon (CPC). 395 fresh or frozen serum and plasma samples from 377 patients were collected and characterized in the laboratory associated to the National HIV Reference Centre of the Rouen University Hospital (CHU) in France. HIV samples routinely tested by 4th generation tests; AxSYM HIV Ag/Ab Combo (Abbott Laboratories) in CPC, and Vidas HIV DUO QUICK (bioMérieux, Marcy l’Etoile, France) in Rouen were re-tested with the ARCHITECT HIV Ag/Ab Combo test kit. Architect HIV Ag/Ab Combo is a chemiluminescent magnetic microparticle-based immunoassay used to determine the presence of HIV-1 p24 antigen and antibody to HIV-1 groups M and O, and HIV-2 by an automated, random access instrument [8]. Sample/Cutoff ratios (S/CO) of >1 were considered reactive and indicated the presence of anti-HIV immunoglobulin and/or p24 antigen; ratios equal to or greater than 0.90 and less than 1.0 were in the gray zone; and ratios of 0.25 indicated the presence of anti-HIV immunoglobulin and/or p24 antigen. For easy representation and comparison of the S/CO with other kits, indexes were calculated relative to the threshold of 1. Samples with discordant results between the “routine” tests and ARCHITECT were analyzed with “local” algorithms in the different laboratories: In Cameroon, samples were repeated in duplicates (ARCHITECT) and further characterized with a 3rd generation test (detection of antibodies only), Genscreen HIV-1/2 version 2 (BIO-RAD, Marnes La Coquette, France) and confirmation assays of western blot (WB) New LAV BLOT 1 and II (BIO-RAD) following the manufacturer’s instructions. Nucleic acid testing (NAT) was performed: briefly, viral RNA was extracted from 1 mL of plasma using the Qiagen viral RNA mini kit (Qiagen, Inc., Chatsworth, CA) as recommended by the manufacturer. The RT (codons 1–255) and Protease genes (codons 1–99) of HIV-1 group M were amplified by Reverse transcriptase PCR using the primers MJ3/MJ4 and 51Prot1/31Prot1, respectively, followed by nested PCR with primers A35/NE1 for RT and 51 Prot 2/31 Prot 2 for protease [15,16]. Visualization of amplified products was done in a 1.5% agarose gel. Discordant samples were tested with an in-house serotyping test as previously described [17], with slight modifications. This inhouse peptide linked immunosorbent assays (PLISA) use synthetic peptides mapping the V3 and gp41/36 regions of HIV (Table 1) to differentiate into the different serotypes (HIV-1: M, O, N, P,
and HIV-2). A first test, “two peptides”, explored the V3 region of HIV-1 groups M (HIV-M) and O (HIV-O). Negative samples in the “two peptides” assay were re-tested in a second PLISA “ten peptides” using peptides mapping the gp41/36 region of HIV-1 groups M, O, N, HIV-2/SIVsm on one hand and V3 peptides of M, O (clade A and consensus), N, P, HIV-2/SIVsm on the other (Table 1). In France, samples were analyzed with (i) 3rd generation kit AxSYM HIV1/2 gO (Abbott), (ii) WB (BIO-RAD), and (iii) Vidas HIV P24 II (bioMérieux) for the determination of the p24 antigen of HIV-1. These tests were performed according to the manufacturer’s instructions, in two consecutive samples collected 15 days apart. Strain diversity was explored on all the positive samples, in Cameroon, using the serotyping assay described above and in France, using the test developed by Simon et al. [18] 4. Results The mean age of the 250 persons tested in Cameroon was 32.4 years (2–68 years) comprising 151 females, 91 males, and 8 children (2–3 years) whose genders were inaccessible. 141/250 (56.4%) had been characterized as HIV positive with AxSYM (Fig. 1a). Of these 141 samples, 126 (89.3%) were also reactive in ARCHITECT with a mean S/CO of 545.34 (range: 1.3–1097.9), and corresponding mean S/CO in AxSYM of 28.7 (range: 1.16–61.88). Fifteen (10.6%) samples were reactive for AxSYM (mean 2.38; range: 1.12–7.63) and non-reactive for ARCHITECT. These 15 discordant samples further tested by Genscreen, WB I and II, NAT and serotyping were nonreactive. On the other hand, 106 of the 109 (97.3%) samples that were non-reactive with AxSYM were also non-reactive with ARCHITECT (Fig. 1a). The remaining 3 (2.7%) ARCHITECT reactive had the following ratios: 10.74, 29.59, and 33.56. These 3 samples were non reactive in Genscreen, WB 1 and II, NAT and serotyping assays. Finally, of the 250 samples tested for both AxSYM and ARCHITECT, 232 (92.8%) were concordant for both assays while 18 (7.2%) were discordant. AxSYM and ARCHITECT misdiagnosed 15 and 3 HIV negative samples respectively. The sensitivity and specificity were 100% (95% CI: 96.3–100%) and 87.9% (95% CI: 80.5–92.8%), respectively for AxSYM, and 100% (95% CI: 96.3–100%) and 97.6% (95% CI: 92.6–99.3), respectively, for ARCHITECT. The mean age of the 377 persons tested in France was 40.7 years (1–86 years) comprising 148 females, 226 males, and 3 persons whose genders were inaccessible. 268/395 samples (67.8%) had been characterized as HIV positive in Vidas (relative index >1) (Fig. 1b). Among these 268 samples, 252 (94%) were also reactive in ARCHITECT with a mean S/CO of 391.79 (range 2.15–852.16), and corresponding mean S/CO of 84.83 (range 4.24–128) in Vidas. Sixteen samples (6%) were reactive for Vidas (mean ratio 2.71; range 1.12–14.72) and non-reactive for ARCHITECT. These discordant samples were further tested with the algorithm defined in CHU Rouen, repeated 2 weeks later on new samples, and all were negative indicating false positive reactions.
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a
250 Samples 141 Reacve AxSYM
126 Reacve Architect
109 Non-Reacve AxSYM 15 Non-Reacve Architect
106 Non-Reacve Architect
3 Reacve Architect
negave with all complementary tools
negave with all complementary tools
b 395 Samples 268 Reacve Vidas 252 Reacve Architect
127 Non reacve Vidas 16 Non reacve Architect
125 Non reacve Architect
2 Reacve Architect
negave with all complementary tools
negave with all complementary tools* for one not enough material
Fig. 1. (a) Comparison of sample reactivity between AxSYM and ARCHITECT in Cameroon. (b) Comparison of sample reactivity between Vidas and ARCHITECT in France.
Of the 127 samples negative with Vidas, 125 were also non-reactive with ARCHITECT (98.4%) (Fig. 1b). The two (1.6%) ARCHITECT reactive samples had the following ratios: 3.22 and 2.44. For one, complementary test (AxSYM HIV1/2 gO) was negative and also negative for the sample collected 2 weeks later. No further tests were carried out for the second due to insufficient sample. Finally, of the 395 samples tested for both Vidas and ARCHITECT, 377 (95.4%) were concordant while 18 (4.5%) were discordant. The sensitivity and specificity were 100% (95% CI: 98.1–100%) and
88.8% (95% CI: 82.2–93.2%) respectively for Vidas, and 100% (95% CI: 98.1–100%) and 98.6% (95% CI: 94.5–99.7%) respectively, for ARCHITECT. In Cameroon, among the 126 samples positive for both AxSYM and ARCHITECT, 110 (87.3%) were serotyped as HIV-M, and 11 (8.7%) as HIV-O (Table 2). Three other samples presented dual reactivity in groups M and O. Finally two samples, with low S/CO values of 6.66 and 3.65 for AxSYM, and 1.3 and 5.54 for ARCHITECT, were negative for serotyping. Because of sample exhaustion no further molecular tests were carried out. In France, the 252 positive
Table 2 Cutoff ratios of the different samples from Cameroon and France, in the three tests.
Group M, Cameroon (n = 110) Group M, France (n = 216) Group O, Cameroon (n = 11) Group O, France (n = 23) Group M + O, Cameroon (n = 3) Group N, France (n = 1) Group P, France (n = 1) HIV 2, France (n = 9) HIV 1 + 2, France (n = 2)
AxSYM HIV Ag/Ab combo (Abbott)
ARCHITECT HIV Ag/Ab Combo (Abbott)
Vidas HIV DUO Quick (bioMérieux)
Mean ratio
Maximum ratio
Minimum ratio
Mean ratio
Maximum ratio
Minimum ratio
Mean ratio
Maximum ratio
Minimum ratio
28.98
61.88
2.98
490.84
1097.92
5.94
/
/
/
/
/
/
438.60
852.16
8.65
85.71
128
6.04
14.44
30.75
5.74
107.92
382.6
7.73
/
/
/
/
/
/
40.78
224.97
2.15
76.96
92.68
4.24
19.43
27.42
18.38
521.43
808.24
14.89
/
/
/
/
/
/
NA (21.42)
NA
NA
NA (16.92)
NA
NA
/
/
/
NA
NA
NA (52.2)
NA
NA
/ /
/ /
/ /
NA (537.95) 118.26 NA (712.83 and 719.36)
132.01 NA
94.87 NA
92.53 NA (92.64 and 99.68)
101.36 NA
85.36 NA
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Fig. 2. Logarithmic representation of HIV-1 groups M, O and M/O reactivities ratio for samples from Cameroon and France.
samples for both Vidas and ARCHITECT provided a representative diversity of strains circulating in the country defined by the following serotypes (Table 2): HIV-M (n = 216; 214 patients), HIV-O (n = 23; 22 patients), HIV-N (n = 1), HIV-P (n = 1), HIV-2 (n = 9), and two samples HIV-1 + HIV-2 dual reactive (n = 2). The samples positive with ARCHITECT presented different values according to the serogroups (Table 2, Fig. 2). HIV-M values were concentrated toward the high range of 100–1000 (Fig. 2), both in Cameroon (mean ratio of 490.84) and France (mean ratio of 438.60) (Table 2). The HIV-O had lower mean ratios (107.92 and 40.78 for Cameroon and France respectively) (Table 2), and are more widespread with maximum ratio not above 400 (Fig. 2). These results indicate a clear difference of reactivity between these two groups. Similar observations were made for the lower HIV-2 mean ratio of 118.26 compared to those of HIV-M. Whenever there is dual reactivity, HIV-M + O or HIV-1 + 2, the sample reactivity is comparable to HIV-M (Table 2). The lone HIV-P sample (derived from SIV infecting gorilla [19]), reacted as HIV-M despite its genetic links with group O. The weak reactivity of HIV-N, could be associated to the acute nature of the sample (seroconversion phase) rather than its antigenic divergence [20]. Of note, AxSYM values are generally lower than those with ARCHITECT, with a lower gradient of reactivity, but similar reactivity between HIV-M and O (Table 2, Fig. 2). The reactivities obtained with the Vidas are more concentrated irrespective of genetic diversity. These results indicate that ARCHITECT reactivity allows the “suspicion” of the serotype/serogroup of the infecting strains, better than AxSYM and Vidas that are more homogeneous. 5. Discussion Several studies have evaluated ARCHITECT in different geographical locations with different HIV prevalence [4,7,21–25]. Here, we conducted a large field evaluation on 645 samples representing
various clinical situations from populations living in sub-Saharan Africa and Europe, permitting a direct comparison of the results. Moreover, this panel is representative to a great extent of the HIV genetic diversity, including distinct variants that are circulating in Europe and in Central Africa, with the rare variants of groups N and P. The results show that the ARCHITECT Combo assay correctly detected all HIV positive samples, whatever the type of population and the clinical stage. This high sensitivity concur previous observations [7,22] highlighting its usefulness in determining established and recent infections [23,25]. Furthermore the presence of antigens representative of HIV-1 group M, O and HIV-2 allows for the detection of a large specter of HIV antibodies even those directed toward the rare groups of HIV-1 group N and P, thereby limiting false negative results in environments of high genetic variability. Our findings therefore complement the evaluations that have been done by others [7,8,22,26,27]. S/CO ratios observed with ARCHITECT are different following the serotype/serogroup, with generally higher ratios compared to AxSYM and Vidas. The absence of a complete correlation between ratios and HIV variants do not allow for the identification of the serotype/serogroup based on the S/CO values only, nevertheless these values could be very informative. An important characteristic of group O samples was their comparatively low and heterogeneous ratios (2.15–382.6), compared to group M ratios; this was also observed for HIV-2 but with a lower ratio (around 100) and a lower widespread distribution. These lower values could highlight the divergence at the antigenic level between group O, HIV-2 and group M. The heterogeneous values observed for HIV-O could be explained by the genetic diversity found among this group particularly at the immunodominant epitope region of the envelope protein, which serves as a target for detection [28,29]. As a consequence of the widespread S/CO ratios observed with diverse strains, the classical S/CO values observed for false positive
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samples could be misleading. In a previous study, in a low risk population [30], the S/CO of false-positive results with ARCHITECT was 2.94 (range: 1.00–34.59). It was also observed that false positive reactivity was higher when this ratio of ARCHITECT was in the low range of 1–5 [23]. Here, we observed a higher mean S/CO of 15.9 (2.44–33.56) for false positive samples from both Cameroon and France. These values are generally lower than the values obtained for HIV-1 group M samples, but closer to values of strains such as HIV-1 group O (lowest ratio of 2.15), which could be wrongly taken for a false positive reactivity. Thus, such low values need complementary analyses to be performed particularly in regions with high HIV genetic diversity, which could definitely distinguish HIV variants from false positive reactivity. Diagnostic laboratories therefore will need to adapt to these new ranges of values and take a lot of caution when interpreting test results. Our results show an improvement in the specificities of ARCHITECT (97.6% and 98.6% for Cameroon and France respectively) compared to AxSYM (87.9%) and Vidas (88.8%). It should nevertheless be noted that these results were obtained from samples preselected by these two tests, which could introduce some bias to the analysis. However, we corroborate earlier observations where ARCHITECT was shown to have a higher specificity than AxSym [8] or other tests [4,7]. The specificities in Cameroon (97.6%) fall short of the WHO recommendations of specificities of 98% [31]. In Africa, endemic infections affect the performances of HIV diagnostic tests and particularly the specificities as a result of cross reactivity [32]. Low specificities with fourth-generation tests have been reported in Cameroon, Tanzania and Uganda [27,33] and could suggest some peculiarities involved with HIV diagnosis in these populations. A fourth generation HIV assay, Abbott Murex HIV antigen/antibody EIA, has been particularly shown in Tanzanian adolescents to have a very low specificity of 91.5% [33]. However, the specificities observed in Cameroon with ARCHITECT was not very different from that observed in France (97.6 versus 98.6%). This could be due to some samples tested in the French site, from persons of African origin living in France. Our results further highlight the fact that serological tests for HIV may perform differently in different populations. ARCHITECT, with a marked improvement in specificity compared to AxSYM and Vidas, should reduce the number of false positive samples, and thereby evade the inconveniences of such results, i.e. increased anxiety for persons with false positive results and the need for other more direct tests such as NAT and/or ELISAs/WB for confirmation, which are costly and time consuming. 6. Conclusion This study shows that the ARCHITECT HIV Ag/Ab Combo assay presents an excellent sensitivity. The evaluation on a large panel of samples from various clinical stages and representative of the high HIV genetic diversity, argues for high performances of clinical sensitivity. However, the widespread distribution of ratios for positive samples, particularly with divergent HIV strains, could cover the range of values for false positive samples. Caution is therefore needed in the interpretation of very low values, which could be in favor of an atypical reactivity corresponding to either HIV variant, an acute infection or false positive reactivity. Our study thus promotes the need to evaluate HIV diagnostic tests before introduction into routine diagnostic services, both in the North as well as in the South, where other confounding factors could be involved. Competing interests The authors declare that they have no competing interests.
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