Journal of Clinical Virology 61 (2014) 166–169

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Evaluation in a clinical setting of the performances of a new rapid confirmatory assay for HIV1/2 serodiagnosis I. Abbate a, * , C. Pergola a , M. Pisciotta a , R. Sciamanna a , C. Sias a , N. Orchi b , R. Libertone c , G. Ippolito d , M.R. Capobianchi a a

Laboratory of Virology, INMI, L. Spallanzani, Rome, Italy CRAIDS and Clinical Department, INMI, L. Spallanzani, Rome, Italy Clinical Department, INMI, L. Spallanzani, Rome, Italy d Scientific Direction, INMI, L. Spallanzani, Rome, Italy b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 March 2014 Received in revised form 12 June 2014 Accepted 14 June 2014

Background and objectives: The performances of the new Geenius rapid confirmatory test (Bio-Rad) were evaluated with emphasis towards identifying acute infection (AHI) and discriminating HIV-1/2 in a clinical setting Study design: Serum samples from individuals attending the L. Spallanzani Institute in Rome, Italy, for HIV diagnosis (one year retrospective collection), repeatedly reactive at 4th generation HIV-1/2 screening assays, confirmed with HIV-1 and HIV-2 Western blot (New LAV I and II Bio-Rad), were retested with Geenius. Results: Of 6,200 samples, 406 resulted repeatedly reactive at screening, including samples from clinically confirmed AHI. New LAV I identified 378 HIV-1-positive samples. Of these, Geenius found 377 HIV-1positive and one unclassified HIV-positive. New LAV I classified as indeterminate 18 samples, including 14 from AHI. Among these 14, Geenius results were: 12 positive, 1 indeterminate and 1 negative. Of the remaining, 2 resulted Geenius negative (false-positive screening results) and 2 HIV-2. Ten samples were New LAV I-negative (5 AHI). Geenius results were: 1 (AHI) positive and 9 negative. Geenius detected 110 additional positive samples with no p31 reactivity with respect to New LAV I, with an almost similar prevalence of low avidity samples. Geenius confirmed 3 out of 4 HIV-2 infections identified by New LAV II (one coinfected with HIV-1), while rated as HIV-1 the remaining sample, classified as coinfection by New LAV I and II. Conclusions: Geenius allows fast, sensitive and accurate confirmation of HIV serodiagnosis, including AHI and HIV-2 infections. The high sensitivity, in particular towards AHI, could avoid additional sampling and molecular tests. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Acute infection HIV Diagnosis Western blot Avidity

Background New diagnostic strategies able to allow earlier and more accurate identification of HIV infections may improve treatment outcome and focus prevention efforts [1,2]. Confirmation of HIV

Abbreviations: AHI, acute HIV infection; TAT, turnaround time; WB, western blot; NNRTI, non-nucleoside reverse-transcriptase inhibitors; PI, proteinase inhibitor; EIA, enzyme immunoassay; WHO, World Health Organization; CDC, centers for disease control and prevention. * Corresponding author at: National Institute of Infectious Diseases INMI, Lazzaro Spallanzani, Via Portuense, 292- 00149, Rome, Italy. Tel.: +39 655170655; fax.: +39 65582346. E-mail address: [email protected] (I. Abbate). http://dx.doi.org/10.1016/j.jcv.2014.06.015 1386-6532/ ã 2014 Elsevier B.V. All rights reserved.

serodiagnosis has been performed for more than 20 years with labour-intensive Western blot (WB) techniques [3], but there is a recognised necessity to speed the turnaround time (TAT) for this step. Recent revisions of HIV diagnostic algorithms highlighted the challenge to identify acute HIV infection (AHI) and HIV-2 [4–6]. AHI is characterized by positive HIV nucleic acid testing and negative/indeterminate WB [7]. HIV-2 is not considered a global public health problem, since it remained mainly confined to West Africa, India and Brazil [8]. However, in an increasingly globalized world, migration and population mobility may demand for new approaches for a correct diagnosis. Currently used HIV WB kits are prone to HIV-1/2 misclassification [9,10], with possibly serious consequences, since HIV-2 is naturally resistant to all NNRTI and to some PI, frequently administrated as first line therapy [11,12].

I. Abbate et al. / Journal of Clinical Virology 61 (2014) 166–169

Objectives Objective of the study was to evaluate the performances of the new rapid confirmatory test (Geenius HIV 1/2, Bio-Rad) versus standard WB (New LAV I and II, Bio-Rad) in a clinical setting, with emphasis towards identification of AHI and ability to discriminate HIV-1 and HIV-2. Study design HIV serodiagnosis is performed at the Laboratory of Virology of the National Institute for Infectious Diseases (INMI) L. Spallanzani, Rome, Italy, according to the Italian guidelines [13]. Serum samples reactive to a 4th generation HIV-1/2 assay (ARCHITECT1 HIV Ag/Ab Combo, Abbott Diagnostics, Abbott Park, IL) were retested with another 4th generation assay (VIDAS DUO HIV Ultra, Biomérieux, Marcy-l’Etoile, France); repeatedly reactive samples underwent HIV-1 WB (New LAV I Bio-Rad Redmond, VA), and were classified as positive if there were at least two env reactivity, according to WHO criteria [14]. In case of borderline or low-positive screening results, or in case cases of suspected acute AHI, additional tests (p24 EIA and/or molecular assays) were performed. HIV-2-specific WB (New LAV II, Bio-Rad) was performed when HIV-1 WB was indeterminate without env reactivity, or upon epidemiological evaluation. Serum samples from a one-year retrospective collection, repeatedly reactive to screening assays, that underwent confirmatory testing with WB New LAV I or II, were retested with Geenius. AHI were classified according to Fiebig staging [15]. Fiebig stage I: HIV RNA assay positive; stage II: RNA and p24 antigen tests positive, antibody EIA non-reactive; stage III: RNA, p24 antigen and HIV IgM-sensitive EIA reactive, but Western blot negative; stage IV: as stage III, but in addition indeterminate Western blot pattern; stage V: as stage IV, but reactive Western blot pattern, with no p31 (pol) reactivity; stage VI: as stage V, but full Western blot reactivity including a p31 band. Avidity test was performed, as described in [16,17], on confirmed positive samples lacking p31 reactivity with the ARCHITECT1 HIV Ag/Ab Combo test. Briefly, two aliquots for each serum sample were prepared by a 1:10 dilution with phosphate-buffered saline (PBS) and with 1 m of guanidine (G) as a denaturing agent. The Avidity Index (AI) of HIV antibodies was calculated using the following formula: AI = (S/CO of the G aliquot)/ (S/CO of the PBS aliquot) where S/CO is the sample/cutoff. The AI is expected to be lower than 1 in the initial period after infection and to approach 1 as the antibodies mature. AI values lower than 0.80

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suggest recent HIV infections (i.e., within 6 months of seroconversion). As reference for accuracy of the diagnostic algorithm, both clinical evaluation and the results of subsequent testing were considered; for each acute infection, the institutional testing protocol included collection of sequential samples to establish sero-conversion. Cases with no evidence of sero-conversion, and without other signs of HIV infection were considered false positive at screening. Statistical significance was evaluated by Mann–Whitney and Fisher exact tests, as appropriate. Results Six thousand and two hundred samples, including 24 samples from 16 patients with AHI (5 Fiebig II/III and 11 Fiebig IV with New Lav I ) , were analyzed. Of these, 423 resulted Architect reactive (median S/Co 596.30; Inter Quartile Range, IQR 299.2–872.9). Seventeen (median S/Co 2.81, IQR 1.46–5.77) were not reactive with VIDAS Ultra and resulted false positives at the follow-up. Median S/Co of AHI samples was 25.08 (IQR 16.80–47.84), lower than that observed in all positive samples, but higher than in false positives (p < 0.0001 for both). Samples reactive to both EIA (n = 406) were used to establish the performance of Geenius versus New LAV. The results are reported in Fig. 1. Among the 378 samples HIV-1-positive with New LAV I, 377 resulted HIV-1-positive with Geenius and one was unclassified because reactive to both HIV-1 and HIV-2 env , suggesting co-infection. In this case, HIV-1 infection was confirmed by HIV-RNA testing, but, unfortunately, HIV-2 presence could not be assessed by molecular testing retrospectively, since no residual sample was available for the analysis. Eighteen samples resulted indeterminate with New LAV I, including 14 HIV-1 AHI. Among this latter group, Geenius results were: 12 HIV-1 positives, 1 indeterminate and 1 negative. For the remaining 4, Geenius results were: 2 negatives (false-positive results) and 2 HIV-2. For the remaining 10 negative samples to New LAV I (including 5 HIV-1 AHI), Geenius results were: 1 (AHI) positive and 9 negatives. From these results, considering WHO criteria for WB reactivity, Geenius reclassified 16 samples: 1 from negative to positive, 12 from indeterminate to positive and 3 from indeterminate to negative. In Table 1, the single antibody reactivity detected in the AHI samples, displaying discordant results between New LAV I and Geenius are reported. Of note, 10 New LAV Iindeterminate samples on the basis of WHO criteria, reclassified as positive with Genius, should have been considered positive according to the less stringent criteria of the Consortium for

Fig. 1. Schematic representation of the study results, obtained by comparing Geenius assay with New LAV I and New Lav II, for confirmation of HIV serodiagnosis in a clinical setting. *FP (false positives).

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I. Abbate et al. / Journal of Clinical Virology 61 (2014) 166–169

Table 1 Single antibody reactivity of AHI samples with discordant confirmatory test results. Sample ID

New LAV I band reactivity

New LAV I classification

Geenius band reactivity

Geenius classification

C3410 C4892 C5370 C5383 C158 C1150 C2816 C3308 C3349 C3394 C3496 C3723 C158 C48

None gp160, p55, p24 gp160, p55, p24 gp160,p66, p55, p52, p24 gp160, p55, p24 p55, p24 gp160, p66, p55, p24 gp160, p55, p24 gp160, p55, p24 p24 gp160, p55, p24 gp160, p55, p24 gp160, p55, p24 p55

Negative Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate Indeterminate

HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 HIV-1 none

HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 Positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV-1 positive HIV negative

Retrovirus Serology Standardization (at least one env reactivity with a simultaneous reactivity for one gag or one pol product) [18]. Considering p31 reactivity, 23 out of 378 HIV-1 confirmed positive samples were p31-negative (possible Fiebig V) with both New LAV I and Geenius; a significantly higher number of samples resulted p31-negative with Geenius (133, including the 23 p31-negative by New LAV I). The avidity test, performed on all 133 p31-negative samples, resulted low (

2 serodiagnosis.

The performances of the new Geenius rapid confirmatory test (Bio-Rad) were evaluated with emphasis towards identifying acute infection (AHI) and discr...
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