Virological exploration of individuals with discordant HIV screening tests

Journal of Clinical Virology 30 (2004) 218–223

Virological exploration of individuals with discordant HIV screening tests C. Pasquier a,∗ , K. Sandres-Sauné a , J.-M. Mansuy a , B. Puissant b , R. Viraben c , N. Spenato c , M. Miédougé a , P. Massip d , A. Blancher b , J. Izopet a a

Laboratoire de Virologie, variabilité virale, EA2046-IFR30, place du Dr. Baylac, Hˆopital Purpan, TSA 40031, F-31 059 Toulouse Cedex, France b Laboratoire d’Immunologie, F-31 059 Toulouse Cedex, France c Centre de dépistage Anonyme et Gratuit F-31 059 Toulouse Cedex, France d Service des Maladies Infectieuses et Tropicales, Hˆ opital Purpan, place du Dr Baylac, TAS40031, F-31059 Toulouse Cedex, France Received 28 July 2003; accepted 22 September 2003

Abstract Screening for HIV infection can use many algorithms. When two different HIV antibody assays are used, discordant results may occur. To discriminate between HIV seroconversion, HIV variant infection and false positive reactivity, 30 consecutive subjects with two discordant HIV antibody-screening assays were extensively investigated for HIV infection. No subject had HIV seroconversion or reached HIV seropositivity criteria after a follow-up of 3 months. By contrast 36% became HIV negative by the use of both HIV screening assays. p24 Antigen, HIV-1 RNA, HIV-1 DNA, HIV-2 DNA assays and HIV isolation by sensitive culture were unable to identify HIV infection in the 30 subjects with discordant HIV screening assays. The data suggest that the use of two HIV screening assays increase false-positive HIV results without increasing clinical sensitivity. To compliment follow-up of HIV screening, early testing for HIV RNA could be useful to identify or eliminate a recent infection. © 2003 Elsevier B.V. All rights reserved. Keywords: HIV diagnosis; HIV antibodies; HIV-1 RNA; Indeterminate HIV Western blot; HIV screening; HIV false positive test

1. Introduction Many algorithms were proposed for the diagnosis of HIV infection (Centers for Disease Control and Prevention C, 2001; Mylonakis et al., 2000; World Health Organization, 1997). Diagnosis and surveillance strategies proposed by UNAIDS and other organisations are based on the use of first line assays, usually anti-HIV-1 antibody screening tests, and confirmatory assays in case of positive or indeterminate result, usually Western blot or immunoblot. Persons newly diagnosed as HIV seropositive on the basis of their first sample are usually tested on a second sample. In France, screening for anti-HIV antibodies relies on the use of two different assays on each sample (Agence Nationale d’Accréditation et d’Evaluation en Santé, 2000). When both tests are non-reactive, absence of HIV antibodies is concluded. When both tests are reactive or discordant, i.e. non-reactive/reactive, a HIV Western blot has to be ∗ Corresponding author. Tel.: +33-5-61-77-90-18; fax: +33-5-61-77-25-42. E-mail address: [email protected] (C. Pasquier).

1386-6532/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2003.09.018

performed for confirmation of HIV antibodies specificity. Confirmatory assays have to be positive to conclude the presence of HIV antibodies. Discordance between assays can occur leading to interpretation difficulties and high level of anxiety for the tested subject. In adult subjects, discordant HIV screening assays can be observed in three settings. Firstly, HIV discordant screening test can be observed in recent HIV infection (Hecht et al., 2002). Most often, this is a short-term discordance and both assays will give reactive results on a second sample taken a few days up to 3 months later (Busch and Satten, 1997). Secondly, it can be observed for some specimen from patients infected by particular HIV-1 or HIV-2 variants. Some of these variants may not be detected by some HIV antibody screening assays and give indeterminate Western blot results. Thirdly, signal may not be linked to an HIV infection; it is a false positive result (Mylonakis et al., 2000). The follow up of HIV antibodies reactivity usually shows stability or a decrease of the reactivity during time. HIV-1 and HIV-2 Western blots remain negative or indeterminate. To try to give a definitive and rapid interpretation toward HIV infection a multiplicity

C. Pasquier et al. / Journal of Clinical Virology 30 (2004) 218–223

of testing and various HIV complementary tests are often performed. In order to clarify the contribution of each figure, we report here the extensive HIV investigations of 30 consecutive subjects with two discordant HIV antibody screening assays on a first sample, including some subjects with high risk of contracting HIV. They were tested for p24 antigen detection, HIV-1 RNA detection (2 assays), HIV-1 DNA detection (2 assays) and HIV-2 DNA. Virus culture was also performed for several patients using a sensitive technique.

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and that did not reach these criteria were considered as indeterminate. 2.3. P24 antigen detection P24 antigen testing was performed using VIDAS HIV P24 II Tests (BioMérieux Sa, Marcy l’Etoile, France) as recommended by manufacturer. The detection limit of the assay is 5 pg/ml. 2.4. HIV-1 RNA detection

2. Material and methods 2.1. Patients and samples Between January 2000 and June 2001, 30 adult subjects were tested for HIV antibodies with discordant results using two different ELISA assays (Table 1). Subjects were outpatients attending a sexual transmitted diseases clinic and university hospital patients. No subject was involved in HIV vaccination trials. All subjects were asked for a second serum sample at 3 month to identify a potential HIV seroconversion. The second sample was tested for HIV-1 RNA in plasma, HIV-1 proviral DNA in PBMC and partially HIV culture isolation. A total of 19 subjects were women, 11 were men. Their mean age was 37 years with no significant difference according the sex of subject. HIV risk factors were identified for six subjects: regular unprotected sex with a HIV-1 infected partner for patient #4, regular unprotected with multiple partners for patient #9, prostitution in Sierra Leone and France for patient #29 and for patients #14, #27, #30, occasional unprotected sex. Twenty four subjects had no known HIV risk factors. Subjects #7, #17, #25 and #26 were pregnant on the date of first sampling.

Detection of HIV-1 RNA was performed using two different commercially available RT-PCR assays: Cobas Amplicor HIV-1 MonitorTM v1.5 (Roche Molecular Diagnostic, Meylan, France) and Abbott LCxTM HIV RNA quantitative (Abbott Diagnostic, Rungis, France). The HIV-1 MonitorTM assay is based on a gag gene fragment amplification and can only detect HIV-1 group M strains. The HIV-1 LCxTM assay is based on a pol gene fragment amplification and can detect HIV-1 group M and O strains. Limit of detection for both assays is 50 copies/ml. 2.5. HIV-1 DNA detection Detection of HIV-1 proviral DNA was performed using two different PCR assays: HIV-1 AmplicorTM Roche (Roche Molecular Diagnostic, Meylan, France) using add-in primers for HIV-1 group M strains (Bogh et al., 2001) and HPOL PCR (Fransen et al., 1994). HIV-1 AmplicorTM assay is based on a gag gene fragment amplification and can only detect HIV-1 group M strains. HPOL PCR is based on a pol gene fragment amplification and can detect HIV-1 group M and O strains. Limit of detection for both assays is 5 copies/106 PBMC. 2.6. HIV-2 DNA detection

2.2. HIV antibodies For each subject, HIV-1 and HIV-2 antibodies screening was performed on two samples taken at 3 month-interval using both Genscreen Plus (Sanofi-Pasteur BIO-RAD, Marnes la Coquette, France) and Abbott AXSYM HIV 1/2gO (Abbott Diagnostic, Rungis, France) assays. Tests were performed as recommended by the manufacturers. The grey-zone was defined as 0.8–1.2 of ratio for Genscreen assay, results above 1.2 were considered as reactive. There was no grey-zone for AXSYM results, results above a ratio of 1 were considered as reactive. HIV-1 and HIV-2 Western blot (NEW LAV BLOT I or II, BIORAD, Marnes la Coquette, France) were performed on first sample taken for reactive or grey-zone screening results. Western blots were interpreted as recommended by the manufacturer: Western blot was considered as positive when two glycoproteins and one other protein band were reactive (WHO, 1991). Samples that had reactive band(s)

Detection of HIV-2 proviral DNA was performed using a real-time PCR protocol on Light-CyclerTM system (Roche Diagnostics Systems) as previously described (Damond et al., 2001; Damond et al., 2002). This assay is based on a gag gene fragment amplification and detects HIV-2 subtypes A and B. Limit of detection for this assay is 5 copies/105 PBMC. 2.7. HIV sensitive culture Blood was collected in CPT (cell preparation tube)-citrate (Becton Dickinson VACUTAINER systems, Franklin Lake, USA) vials. As recommended by the manufacturer, CPT vials were centrifuged to isolate the buffy coat from plasma and other cells. PBMC were collected and washed twice in PBS and cell concentration adjusted to 10 × 106 cells/ml in PBS containing 2% FCS. PBMC were depleted in CD8+ cells using anti-CD8 coated magnetic beads

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Table 1 Demographic data and results of HIV exploration for the 30 subjects with HIV screening discordant test results Age

77 21 76 24 53 27 44 63 63 23 39 39 21 27 17 38 41 43 57 16 18 66 23 43 32 25 39 27 18 18

F F F M F M F M M F F F F M F F F F M F F F M M F F M M F M

HIV antibody screening tests

Western blot

M0 Test

HIV-1

HIV-2

Ag p24

Monitor

LCX

Amplicor

HPOL

p31(±) – – – – – p24(±) – – – – p17(±) p51(±) p24(±) – p31(±), p24(±) – – – p31(±) p66(±),p24(±) p55(+) – p17(±) p31(±) – p31(±), p24(±) – p55(±), p24(±), p17(±) p24(±)

p26(+) – p26(±) – – – p26(±) – – p26(±) – p16(±) p26(±) p16(±) – p26(±) – – – – – p26(±) – – – p26(±) – – p26(±) p68(±), p16(+)

– – NT – – – NT NT – – – – – – – – – – – NT – – – – – NT – – NT –

– – NT – – – NT NT – – – – – – – – – – – NT – – – – – – NT – NT –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – NT – – – – – – – – – – – – – – – – – – – – – – – – –

M3 Test

A

B

A

B

1.5 8.2 0.3 0.2 0.2 0.2 0.9 1.6 1.5 0.1 0.3 11.3 0.3 4.7 0.2 1.0 1.6 11.5 0.2 5.5 0.2 2.3 0.1 4.7 5.7 4.4 3.9 1.4 5.4 0.9

0.3 0.3 4.3 1.3 1.4 1.6 0.4 0.3 0.4 2.3 5.5 0.3 1.1 0.3 3.6 0.3 0.3 0.4 6.6 0.4 2.0 0.3 5.2 0.3 0.4 0.3 0.4 0.3 0.3 0.4

0.4 2.6 0.2 0.2 0.2 0.3 0.5 1.4 1.4 0.3 0.2 11.2 0.3 1.4 0.1 3.8 0.0 22.7 0.2 0.3 0.2 2.2 0.2 9.1 1.0 1.7 13.4 0.6 2.9 0.8

0.3 0.4 0.5 1.1 1.0 0.3 0.4 0.5 0.5 1.1 8.5 0.4 0.8 0.4 4.3 0.4 0.3 0.3 6.3 0.5 1.7 0.3 4.6 0.4 0.3 0.4 0.3 0.3 0.3 0.3

HIV-1 RNA

HIV-1 DNA

HIV-2 DNA

Culture

– NT – – NT – – – – – NT – – – – – – – – – NT – NT – – – – NT NT –

NT NT NT – – NT NT NT – NT – – – NT NT NT NT NT – NT – NT – NT NT NT NT – NT NT

For each subject, age at discovery of discordant HIV screening, gender (M for male and F for female), ratios of screening assay performed on first and second samples by two assays (A: GenscreenTM HIV and B: AxsymTM HIV), HIV-1 and HIV-2 Western blot patterns performed on the first sample and results of HIV-1 and HIV-2 direct markers and HIV culture are presented.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Gender

C. Pasquier et al. / Journal of Clinical Virology 30 (2004) 218–223

(Dynabeads M450 CD8 T, DYNAL Biotech, Compiègne, France) as recommended by the manufacturer. For each sample, a total of 5 × 106 CD8-depleted PBMC per well was incubated in a 24-well plaque in medium (RPMI 1640 Medium containing 20% FCS, penicillin-streptomycin, 4 IU/ml interleukin (IL)-2 (Roche Molecular biochemicals, Meylan, France) at 37 ◦ C with 5% CO2 . To induce activation of resting lymphocytes 2.5 ␮g/ml of anti-CD28, 10 ␮g/ml of antiCD3 (Beckman Coulter, Villepinte Roissy-CDG, France) were added. Half of the medium was replaced three times a week during the two weeks of culture follow up. Detection of produced HIV-1 was performed on day 7 and 14 using AmplicorTM HIV-1 MonitorTM assay on 500 ␮l of culture supernatant and RT activity sensitive detection using F-PERT (Lovatt et al., 1999). In addition, HIV-1 proviral DNA amplification using HPOL nested PCR on 1 × 106 cells harvested at day 14 was performed. On a panel of 13 HIV-1 infected patients with a median of 19 RNA copies/ml (range 3–68,600), the sensitivity of this virus culture was 100%. This culture protocol was also tested on 3 HIV-2 infected subjects with positive results.

3. Results 3.1. HIV antibodies assays As shown in Table 1, all subjects displayed discordant results after anti-HIV testing on the first sample taken, i.e. one negative result and the other one positive or grey-zone. GenscreenTM HIV assay gave 16 reactive results, 3 indeterminate results and 11 negative results. The median ratios (s/co quotient) were 4.55 (range 1.4–11.5) for positive samples and 0.2 (range 0.1–0.3) for negative samples. AxsymTM HIV assay gave 10 reactive results, 1 indeterminate result and 19 negative results. The median ratios were 3.38 (range 1.3–6.6) for positive samples and 0.3 (range 0.3–0.4) for negative samples. There was no significant difference of ratios for both tests according to the gender of subjects. Among these 30 subjects, 9 (30%) had antibody HIV screening that became negative on the 3-month control and a total of 11 (36%) thereafter during follow-up. Among them, subject #7 had delivery between the two samplings. Among the 16 reactive results using GenscreenTM assay, all were controlled on at least one following sample and 4 were found non-reactive and 1 was in the in grey-zone using the same assay. Among the 10 reactive results using AxsymTM assay, 2 were found non-reactive and 3 in grey-zone using the same assay on a subsequent repeat sample. All specimens were tested by HIV-1 and HIV-2 Western blot on their first discordant sample (Table 1). A total of 13 samples gave negative HIV-1 and HIV-2 Western blots. A total of 16 samples had negative HIV-1 Western blot, none were positive. The 14 HIV-1 indeterminate patterns

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had reactivity toward core or matrix proteins, i.e. p17, p24, p31, p51, p55 or p66. A total of 18 samples had negative HIV-2 Western blot, none were positive. The 12 HIV-2 indeterminate patterns had reactivity toward core or matrix proteins, i.e. p16 or p26. In summary, during the 3 month-lag of testing or thereafter no subject had HIV seroconversion or reached anti-HIV seropositivity criteria. By contrast 11 subjects (36%) became anti-HIV negative by the use of both HIV screening assays. 3.2. HIV direct assays Results of HIV-1 genomic detections performed on the second sample taken are summarized in Table 1. P24 antigen was analyzed in serum using VIDAS assay. A total of 24 samples were tested and all of them gave negative results. LCXTM assay was performed in 30 samples with no HIV-1 RNA detected. Using AmplicorTM HIV-1 MonitorTM v1.5 ultra sensitive assay as second technique gave in 30 samples no HIV-1 RNA was detected. HIV-1 DNA detection in PBMC was performed using two different assays. HIV-1 AmplicorTM with add-in primers gave 30 negative results on 30 samples tested. HPOL nested PCR gave 29 negative results on 29 samples tested. HIV-2 DNA detection in PBMC was performed on 23 samples and gave 23 negative results. A total of 10 samples was subjected to HIV sensitive culture. Results were negative in all cases after 15 days of culture as reported in Table 1. In summary, HIV direct assays were unable to identify HIV infection in the 30 subjects with discordant HIV screening assays.

4. Discussion The use of two different assays for HIV antibody screening, as performed in France, was introduced to avoid the release of false negative anti-HIV screening results. This practice leads to discordant results that represent around 1.26% of samples routinely tested for HIV in our laboratory. In most cases, further virological explorations and subsequent controls over time will be needed to try to give a definitive interpretation on HIV status. The subject with discordant HIV results might then have to live with anxiety generated by his ambiguious HIV status. No ongoing HIV infection was identified among the 30 subjects having discordant HIV screening tests. Presence of high risk factors or confirmed exposure to HIV was reported for 6 subjects in this study, but no diagnosis of HIV infection could be made. In HIV-1 exposed–non-infected subjects, genetic factors such as CCR5 polymorphism can be involved in the absence of infection. None of our 30 patients was 32 CCR5 homozygous (data not shown).

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To detect HIV-1 variant we used HPOL PCR that is known to amplify HIV-1 group M and numerous HIV-1 group O strains (Bogh et al., 2001). HIV-1 group O strains are known to give poor reactivity with HIV screening test and indeterminate Western blot (Loussert-Ajaka et al., 1995; Mauclere et al., 1997). To detect HIV-2 DNA, we have performed a homemade PCR developed for HIV-2 encountered in France (Damond et al., 2001). To be extensive in exploration for HIV variants, HIV culture was performed for 10 subjects. The use of HIV sensitive culture allowed us to induce HIV production from latently infected cell and to multiply the virus in analogous cells. This culture protocol is highly sensitive and can therefore identify virus replication in 77% of infected patients with undetectable HIV-1 RNA in plasma (<50 copies/ml). Because of its independence of HIV genomic sequence, RT activity detection in culture supernatant was performed to then detect virus production. The systematic detection of HIV variants is therefore labour intensive and should probably be limited to subjects with clinical or immunological signs of HIV infection and not performed in a laboratory routine setting. Performance of HIV antibody tests has improved over the years. They are now highly sensitive, and HIV seroconversion is detected earlier (Weber et al., 1995). By contrast to higher sensitivity, specificity of HIV screening assays might be lower leading to false positive results. In ongoing primary HIV infection, markers such as anti-HIV screening test and Western blot should become reactive during the follow up of subjects. The delay observed before reactivity for HIV screening test after infection is usually between 3 and 12 weeks (Gurtler et al., 1998; Hecht et al., 2002), thus both HIV assays should become rapidly positive after observation of a screening result test discordance. Western blots remained undeterminate in all cases because of their lower sensitivity compared to screening assay. In this setting, Western blots were unable to discriminate between false reactivity and HIV seroconversion because the observed profiles were often compatible with both possibilities. In contrast, HIV antibody screening tests that combine p24 antigen detection are useful for early diagnosis (Gurtler et al., 1998; Ly et al., 2001; Urnovitz et al., 1997; Weber, 2003). In this setting, the use of direct markers such as HIV-1 RNA viral load is more informative (Kleinman et al., 1998; Mylonakis et al., 2000) and is able to identify ongoing HIV-1 infection before or after HIV seroconversion (Pilcher et al., 2002). All HIV-1 direct markers tested in this study remained negative. These HIV indeterminate results revealed no evidence of HIV infection in a 3 months follow-up or afterwards as shown for HIV indeterminate blood donors in long term follow-up (Watt et al., 2000). False positive HIV screening tests are reported in pregnant women (Doran and Parra, 2000), subjects with autoimmune disease (Janvier et al., 1989), renal and liver diseases, intra-venous drug use and probably some vaccines (Celum et al., 1994; Mylonakis

et al., 2000) and infectious diseases (Watt et al., 2000). Pregnancy, alloimmunization and autoimmune disease may explain the higher frequency of HIV false positive results observed in women. This HIV reactivity can then persist over a long period or spontaneously revert to negativity as reported for 34% in this study. The use of two different anti-HIV screening tests was supposed to avoid the release of false negative anti-HIV screening results. In fact, it prevent the release of false positive screening results and facilitated detection of working errors. However, this practice can give frequent discordant results that requires complementary HIV tests being performed. Extensive virological investigations realized in this study remain unsuccessful to identify HIV infection. Discordant anti-HIV screening results remained constant over time or reversed to negative and could then be considered as false positive results. The use of two HIV screening assays probably increase false-positive HIV results without increasing clinical sensitivity. Early testing for HIV RNA or HIV screening tests combining p24 antigen and HIV antibody detection could be more useful to identify or eliminate a recent infection.

References AIDS: proposed WHO criteria for interpreting Western blot assays for HIV-1, HIV-2, and HTLV-I/HTLV-II. Bull World Health Organ 1991; 69:127–9, 131–3. Agence Nationale d’Accréditation et d’Evaluation en Santé. Recommandations pour la pratique clinique : stratégies du diagnostic biologique de l’infection due au VIH chez les sujets aˆ gés de plus de 18 mois (à l’exclusion du dépistage sur dons de sang et chez les donneurs d’organes ou de tissus). http://www.anaes.fr 2000. Bogh M, Machuca R, Gerstoft J, Pedersen C, Obel N, Kvinesdal B, Nielsen H, Nielsen C. Subtype-specific problems with qualitative Amplicor HIV-1 DNA PCR test. J Clin Virol 2001;20:149–53. Busch MP, Satten GA. 1997. Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am J Med 1997;102(117–124):125–6. Celum CL, Coombs RW, Jones M, Murphy V, Fisher L, Grant C, Corey L, Inui T, Wener MH, Holmes KK. Risk factors for repeatedly reactive HIV-1 EIA and indeterminate Western blots. A population-based case-control study. Arch Intern Med 1994;154:1129–37. Centers for Disease Control and Prevention C. Revised guidelines for HIV counseling, testing, and referral. Morbidity and Mortality Weekly Report 2001;50:RR19. Damond F, Apetrei C, Robertson DL, Souquiere S, Lepretre A, Matheron S, Plantier JC, Brun-Vezinet F, Simon F. Variability of human immunodeficiency virus type 2 (HIV-2) infecting patients living in France. Virology 2001;280:19–30. Damond F, Descamps D, Farfara I, Telles JN, Puyeo S, Campa P, Lepretre A, Matheron S, Brun-Vezinet F, Simon F. Quantification of proviral load of human immunodeficiency virus type 2 subtypes A and B using real-time PCR. J Clin Microbiol 2001;39:4264–8. Damond F, Gueudin M, Pueyo S, Farfara I, Robertson DL, Descamps D, Chene G, Matheron S, Campa P, Brun-Vezinet F, Simon F. Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections. J Clin Microbiol 2002;40:3654–9. Doran TI, Parra E. False-positive and indeterminate human immunodeficiency virus test results in pregnant women. Arch Fam Med 2000;9:924–9.

C. Pasquier et al. / Journal of Clinical Virology 30 (2004) 218–223 Fransen K, Zhong P, De Beenhouwer H, Carpels G, Peeters M, Louwagie J, Janssens W, Piot P, van der Groen G. Design and evaluation of new, highly sensitive and specific primers for polymerase chain reaction detection of HIV-1 infected primary lymphocytes. Mol Cell Probes 1994;8:317–22. Gurtler L, Muhlbacher A, Michl U, Hofmann H, Paggi GG, Bossi V, Thorstensson R, R GV, Eiras A, Hernandez JM, Melchior W, Donie F, Weber B. Reduction of the diagnostic window with a new combined p24 antigen and human immunodeficiency virus antibody screening assay. J Virol Methods 1998;75:27–38. Hecht FM, Busch MP, Rawal B, Webb M, Rosenberg E, Swanson M, Chesney M, Anderson J, Levy J, Kahn JO. Use of laboratory tests and clinical symptoms for identification of primary HIV infection. AIDS 2002;16:1119–29. Janvier D, Reviron M, Reviron J. False negative results in a competitive immunoassay for anti-HIV-antibodies due to the presence of rheumatoid factors. Vox Sang 1989;56:237–42. Kleinman S, Busch MP, Hall L, Thomson R, Glynn S, Gallahan D, Ownby HE, Williams AE. False-positive HIV-1 test results in a low-risk screening setting of voluntary blood donation. Retrovirus epidemiology donor study. JAMA 1998;280:1080–5. Loussert-Ajaka I, Chaix ML, Korber B, Letourneur F, Gomas E, Allen E, Ly TD, Brun-Vezinet F, Simon F, Saragosti S. Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France. J Virol 1995;69:5640–9. Lovatt A, Black J, Galbraith D, Doherty I, Moran MW, Shepherd AJ, Griffen A, Bailey A, Wilson N, Smith KT. High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods. J Virol Methods 1999;82: 185–200.

223

Ly TD, Laperche S, Couroucé AM. Early detection of human immunodeficiency virus infection using third and fourth-generation screening assays. Eur J Clin Microbiol Infect Dis 2001;20:104–10. Mauclere P, Loussert-Ajaka I, Damond F, Fagot P, Souquieres S, Monny Lobe M, Mbopi Keou FX, Barre-Sinoussi F, Saragosti S, Brun-Vezinet F, Simon F. Serological and virological characterization of HIV-1 group O infection in Cameroon. AIDS 1997;11:445–53. Mylonakis E, Paliou M, Greenbough TC, Flaningan TP, Letvin NL, Rich JD. Report of a false-positive HIV test result and the potential use of additional tests in establishing HIV serostatus. Arch Intern Med 2000;160:2386–8. Mylonakis E, Paliou M, Lally M, Flanigan TP, Rich JD. Laboratory testing for infection with the human immunodeficiency virus: established and novel approaches. Am J Med 2000;109:568–76. Pilcher CD, McPherson JT, Leone PA, Smurzynski M, Owen-O’Dowd J, Peace-Brewer AL, Harris J, Hicks CB, Eron JJ, Fiscus SA. Real-time. JAMA 2002;288:216–21. Urnovitz HB, Sturge JC, Gottefried TD. Increased sensisitivity of HIV-1 antibody detection. Nat Med 1997;3:1258. Watt G, Chanbancherd P, Brown AE. Human immunodeficiency virus type 1 test results in patients with malaria and dengue infections. Clin Infect Dis 2000;30:819. Weber B, Moshtaghi-Boronjeni M, Bruner M, Preiser W, Breiner M, Doerr HW. Evaluation of the reliability of 6 current anti-HIV-1/HIV-2 enzyme immunoassays. J Virol Methods 1995;55:97–104. Weber B. HIV seroconversion: performance of combined antigen/antibody assays. AIDS 2003;17:931–3. World Health Organization. Joint United Nations Programme on HIV/AIDS (UNAIDS)-WHO : Revised recommendations for the selection and use of antibody test. Weekly Epidemiological Record 1997;72:81–88.