Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection

BackgroundIn the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing.ObjectivesTo evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection.Study designA series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients > 18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children ≤ 18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay.ResultsMultispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot.ConclusionsMultispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2.     

Identification of early HIV infections using the fourth generation Abbott ARCHITECT HIV Ag/Ab Combo chemiluminescent microparticle immunoassay (CIA) in San Diego County

BackgroundHIV screening assays have gone through several generations of development in an effort to narrow the “window period” of detection. Utilizing a fourth generation HIV screening assay has the potential to detect earlier HIV infection, thus reducing HIV-1 transmission.ObjectiveTo identify acute infections to decrease HIV transmission in San Diego County.Study designSerum specimens were collected from clients seen by multiple submitters in San Diego County. All acceptable specimens were screened using the 4th Gen Combo Assay. Initially reactive specimens were repeated in duplicate and if repeatedly reactive, were confirmed by HIV-1 Immunofluorescent Antibody Assay (IFA).IFA negative/inconclusive specimens were sent for HIV-1 NAT and HIV-2 antibody testing to referral laboratories. BioRad Multispot HIV-1/HIV-2 Rapid Test was also performed on a subset of specimens.ResultsOf 14,559 specimens received in 20 months, 14,517 specimens were tested. Of the 14,517 specimens that were tested, a total of 279 (1.9%) specimens were CIA repeatedly reactive and 240 of the 279 confirmed by HIV-1 IFA. Thirty-nine gave IFA negative/inconclusive result and 30 were further tested for HIV-1 NAT and 36 for HIV-2 antibody. Thirteen specimens were considered false positives by CIA and 17 specimens were classified as acute infections. Eleven of 39 IFA negative/inconclusive specimens were further tested by Multispot. Five of the 11 were positive by Multispot.ConclusionThe fourth generation Abbott ARCHITECT HIV Ag/Ab Combo Assay identified 17 patients who may have been missed by the prior HIV-1 screening assay used at San Diego County Public Health Laboratory.     

Evaluation of supplemental testing with the Multispot HIV-1/HIV-2 Rapid Test and APTIMA HIV-1 RNA Qualitative Assay to resolve specimens with indeterminate or negative HIV-1 Western blots

BackgroundThe use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis.ObjectiveTo determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm.Study designRemnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA).ResultsAccording to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive.ConclusionApplying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.     

Evaluation of the performance of a new automated HIV combination assay

BackgroundMore and more countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies.ObjectiveTo assess the performance of a new HIV combo assay: LIAISON^® XL murex HIV Ab/Ag HT.Study designThe assays were examined with a total of 3090 samples that included 769 selected HIV antibody-negative samples, 1849 unselected HIV samples, 15 HIV-1 p24 Ag reference samples, 90 primary HIV-1 infection (PHI) samples, 167 HIV-1 antibody-positive samples (well characterized of groups M and O), 95 HIV-1 antibody-positive samples and 105 HIV-2 antibody-positive samples.ResultsThe specificity of the LIAISON^® XL murex HIV Ab/Ag HT was 99.7%. The analytical sensitivity of Ag p24 of the LIAISON^® XL murex HIV Ab/Ag HT was 0.58 IU/mL and 9.93 pg/mL when using WHO and French national standards, respectively. All screened HIV subtypes was identified by this assay. Also, 90 PHI specimens were detected by this screening assay.ConclusionThe sensitivity and specificity of the LIAISON^® XL murex HIV Ab/Ag HT assay are high. Hence the assay offers automated high-throughput screening with ability to detect primary infection.     

Use of the Abbott Architect HIV antigen/antibody assay in a low incidence population

BackgroundWith the availability of 4th generation HIV diagnostic tests which are capable of detecting acute infection, Iowa evaluated the 3rd and 4th generation HIV test and compared the performance of these products in a low incidence population.ObjectiveThis study was conducted to evaluate the performance of an HIV antigen/antibody combination (4th generation) assay compared to an EIA 3rd generation assay.Study designOver a 4 month period, 2037 specimens submitted for HIV screening were tested by Bio-Rad GS HIV-1/HIV-2 Plus O EIA and the Abbott Architect i1000SR HIV Ag/Ab Combo. The performance characteristics of sensitivity, specificity, positive predictive value and negative predictive value were determined.ResultsOf the 2037 specimens tested, there were 13 (0.64%) true positives detected. None of the positive specimens were from patients in the acute phase of infection. The Abbott antigen/antibody combo assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.85%, 81.25%, and 100% respectively. The Bio-Rad EIA assay had a sensitivity, specificity, positive-predictive value and negative predictive value of 100%, 99.80%, 76.47% and 100%, respectively. The EIA had four false positive results which tested negative by the antigen/antibody assay and western blot.ConclusionIn a low-incidence state where early infections are less commonly encountered, the EIA assay and the antigen/antibody assay performed with near equivalency. The antigen/antibody assay had one less false positive result. While no patients were detected in the acute stage of infection, the use of the antigen/antibody assay presents the opportunity to detect an infected patient sooner and prevent transmission to others.     

Comparative performance of the Geenius™ HIV-1/HIV-2 supplemental test in Florida's public health testing population

BackgroundThe Centers for Disease Control and Prevention (CDC) published updated guidelines in 2014 for the laboratory diagnosis of HIV in the United States, which recommend use of a supplemental immunoassay (IA) that differentiates HIV-1 from HIV-2 after a repeatedly reactive HIV-1/2 antigen/antibody “Combo” screening test. In October 2014, Bio-Rad Laboratories introduced the FDA-cleared Geenius HIV-1/HIV-2 Supplemental assay and in July 2016, it replaced the Multispot HIV-1/HIV-2 differentiation rapid test as the second test in the HIV diagnostic algorithm.ObjectiveTo compare performance of the new FDA-cleared Bio-Rad Geenius HIV-1/HIV-2 Supplemental assay and the Bio-Rad Multispot HIV-1/HIV-2 differentiation assay for use as the primary supplemental test in the 2014 CDC/APHL HIV Diagnostic Algorithm.Study designTwo sets of specimens were used to assess the performance of Geenius; 340 select retrospective specimens, obtained through routine clinical submissions from individuals seeking HIV serostatus determinations and 10 known HIV-2 antibody reactive specimens provided by Bio-Rad Laboratories. Panels were created and characterized solely by in-house laboratory results. The panels consisted of: algorithm-defined “established HIV-1 infections” (n = 250), “acute HIV-1 infections” (n = 20), “early HIV-1 infections” (n = 10) and “false positive Combo specimens” (n = 60).Results

• •Geenius results for the 250 specimens classified as HIV-1 established infections were HIV-1 Positive (246) or HIV Positive Untypable (4), a 100% concordance with HIV positivity observed with Multispot results (HIV-1 Positive, HIV-2 Negative).
• •Of the 20 HIV-1 algorithm-defined acute infection specimens (Multispot Negative), 85% (17/20) were Geenius HIV-1 Negative and 15% (3/20) HIV-1 Indeterminate.
• •Similarly, of the 10 algorithm-defined early infection specimens (MS Indeterminate), 30% (3/10) were Geenius HIV-1 Indeterminate, 60% (6/10) HIV-1 Positive and 10% (1/10) HIV Negative.
• •Of the 60 false positive HIV-1/2 Combo panel, 98.3% (59/60) were HIV-1 Negative by Geenius and 95% (57/60) were HIV-1 Negative by Multispot.
• •Geenius was concordant with all HIV-2 antibody positive specimens.

ConclusionsThe Geenius assay provides significant advantages over Multispot as an appropriate replacement for the primary supplemental test in the HIV Diagnostic Algorithm. In this retrospective study, Geenius was highly concordant with Multispot, reclassified some acute and early algorithm-defined HIV-1 positive specimens and demonstrated a potential decrease in the number HIV-1 RNA nucleic acid amplification tests needed to complete the diagnostic algorithm.     

The fourth generation AlereTM HIV Combo rapid test improves detection of acute infection in MTN-003 (VOICE) samples

BackgroundEarly and accurate detection of HIV is crucial when using pre-exposure prophylaxis (PrEP) for HIV prevention to avoid PrEP initiation in acutely infected individuals and to minimize the risk of drug resistance in individuals with breakthrough infection.ObjectiveTo determine if fourth-generation antigen/antibody (Ag/Ab) rapid diagnostic tests (RDT) would have detected HIV infection earlier than the third-generation RDT used in MTN-003 (VOICE).Study design5029 VOICE participants were evaluated with third-generation Alere Determine™ HIV-1/2, OraQuick ADVANCE^® Rapid HIV-1/2, Uni-Gold™ Recombigen^® HIV-1/2 and Bio-Rad GS HIV-1/2 + O EIA; and fourth-generation Alere Determine™ HIV-1/2 Ag/Ab Combo, Conformité Européene (CE)-Marked Alere™ HIV Combo and Bio-Rad HIV Combo Ag/Ab EIA. Multispot^®, GS HIV-1 Western Blot (WB) and Geenius™ (Bio-Rad) were also evaluated.ResultsOf 57 antibody-negative pre-seroconversion plasma samples with HIV RNA >20 copies/mL identified, 16 (28%) were reactive by CE-Marked Alere™ HIV Combo (1 Ab; 9 Ag; 6 Ag/Ab reactive) and 4 (7%) by Alere Determine™ HIV-1/2 Ag/Ab Combo (2 Ab; 2 Ag; 0 Ag/Ab reactive) (p = 0.0005). Multispot^® confirmed only 1 of 16 acute infections while WB and Geenius™ confirmed none. GS HIV Combo Ag/Ab EIA identified 27 of 57 (47%) pre-seroconversion RNA-positive samples.ConclusionIn VOICE, 28% of infections missed by current third-generation RDT would have been identified with the use of CE-Marked Alere™ HIV Combo. Geenius™, Multispot^® and WB were all insensitive ( < 10%) in confirming infections detected by fourth-generation assays. An improved diagnostic algorithm that includes a fourth-generation RDT with HIV RNA testing will be essential for efficiently identifying seroconverters on PrEP.     

Performance evaluation of the FDA-approved Determine™ HIV-1/2 Ag/Ab Combo assay using plasma and whole blood specimens

BackgroundThe Determine™ HIV-1/2 Ag/Ab Combo (DC) rapid test can identify HIV-1 infection earlier than rapid antibody-only tests in plasma specimens.ObjectivesWe compared the performance of DC with a laboratory-based antigen/antibody (Ag/Ab) combo assay in plasma and evaluated antigen reactivity in whole blood specimens.Study designWe tested by DC 508 plasma specimens collected in a prospective study and 107 sequential plasma and simulated whole blood specimens from 20 seroconversion panels. Previous results using the ARCHITECT (ARC) Ag/Ab combo assay were compared to DC results. In seroconversion panels, the days from the first HIV1 RNA-positive test to first DC-reactive in plasma and whole blood was compared. McNemar’s and Wilcoxon signed rank tests were used for statistical analysis.ResultsOf 415 HIV-positive samples, ARC detected 396 (95.4%) and DC 337 (81.2%) (p < 0.0001). DC was reactive in 50.0% of ARC-reactive/MS-negative, 78.6% of ARC-reactive/MS-indeterminate, and 99.6% of ARC-reactive/MS-HIV-1-positive or −undifferentiated specimens. DC antigen reactivity was higher among ARC-reactive/MS-negative than MS-indeterminate samples. In 20 HIV-1 seroconversion panels, there was a significant difference between DC reactivity in plasma (91.1%) and whole blood (56.4%) (p < 0.0001). DC with whole blood showed a significant delay in reactivity compared to plasma (p = 0.008).ConclusionsIn plasma, DC was significantly less sensitive than an instrumented laboratory-based Ag/Ab combo assay. DC in plasma was significantly more sensitive compared to whole blood in early HIV-1 infections. With the U.S. laboratory-based diagnostic algorithm, DC as the first step would likely miss a high proportion of HIV-1 infections in early stages of seroconversion.     

HIV misdiagnosis: A root cause analysis leading to improvements in HIV diagnosis and patient care

BackgroundStandard diagnostic testing for HIV infection has traditionally relied on a high sensitivity HIV antibody screening test using an enzyme-linked immunosorbent assay (ELISA) followed by a high specificity antibody confirmatory test such as a Western Blot. Recently several of the screening assays have been enhanced with an ability to identify p24 antigen thereby narrowing the diagnostic window.ObjectivesTo explore the implications of enhanced HIV screening methods that may be leading to HIV misdiagnoses.Study designA patient deemed to be an HIV infected ‘elite controller’ was found to be misdiagnosed when undergoing detailed investigations prior to initiating antiretroviral therapy. A root cause analysis was performed to identify the causative factors of this misdiagnosis. A retrospective review of all “elite controllers” in Alberta, Canada revealed challenges of current HIV testing algorithms.ResultsTechnical and human factors were identified as being causative in this HIV misdiagnosis including (i) high rates of false reactive results on the Abbott ARCHITECT HIV-1&2 COMBO EIA, (ii) human error in reading the initial Western blot, (iii) HIV algorithmic directives in which confirmatory (Western blot) testing was not performed on a repeatedly reactive screen test. The outcome of this analysis identified opportunities for improvement, including implementation of a newly approved (automated) confirmatory assay and improved communication between the clinician and laboratory.ConclusionsHIV testing remains problematic despite significant advances in HIV test performance and algorithm development, presenting new and unexpected issues. Ensuring a high-quality management system including implementation of the latest HIV technologies and algorithms along with human resources and policies are required to minimize the impact of false positive diagnoses, especially in the era of universal screening and ‘test and treat’ recommendations.     

Multi-centre field evaluation of the performance of the Trinity Biotech Uni-Gold HIV 1/2 rapid test as a first-line screening assay for gay and bisexual men compared with 4th generation laboratory immunoassays

BackgroundThe Trinity Biotech Uni-Gold HIV test (Uni-Gold) is often used as a supplementary rapid test in testing algorithms.ObjectiveTo evaluate the operational performance of the Uni-Gold as a first-line screening test among gay and bisexual men (GBM) in a setting where 4th generation HIV laboratory assays are routinely used.Study designWe compared the performance of Uni-Gold with conventional HIV serology conducted in parallel among GBM attending 22 testing sites. Sensitivity was calculated separately for acute and established infection, defined using 4th generation screening Ag/Ab immunoassay (EIA) and Western blot results. Previous HIV testing history and results of supplementary 3rd generation HIV Ab EIA, and p24 antigen EIA were used to further characterise cases of acute infection.ResultsOf 10,793 specimens tested with Uni-Gold and conventional serology, 94 (0.90%, 95%CI:0.70–1.07) were confirmed as HIV-positive by conventional serology, and 37 (39.4%) were classified as acute infection. Uni-Gold sensitivity was 81.9% overall (77/94, 95%CI:72.6–89.1); 56.8% for acute infection (21/37, 95%CI:39.5–72.9) and 98.2% for established infection (56/57, 95%CI:90.6–100.0). Of 17 false non-reactive Uni-Gold results, 16 were acute infections, and of these seven were p24 antigen reactive but antibody negative. Uni-Gold specificity was 99.9% (10,692/10,699, 95%CI:99.9–100.0), PPV was 91.7% (95%CI:83.6–96.6) and NPV was 99.8% (95%CI:99.7–99.9), respectively.ConclusionsIn this population, Uni-Gold had good specificity and sensitivity was high for established infections when compared to 4th generation laboratory assays, however sensitivity was lower in acute infections. Where rapid tests are used in populations with a high proportion of acute infections, additional testing strategies are needed to detect acute infections.     

Performance of serological and molecular tests within acute HIV infection

BackgroundEarly diagnosis of acute HIV infection can be challenging and is critical in regards to early therapeutic decision making.ObjectivesTo evaluate the performance of different HIV tests in detecting early infections.Study designA total of 83 leftover specimens of 61 study participants who seroconverted were used in this sub-study. 35 HIV RNA positive but still seronegative specimens (acute infections) were used for analysis of the sensitivity of the different assays in detecting early infections and 42 HIV RNA and antibody negative specimens were used for specificity analysis.ResultsFour (11%) specimens out of 35 acute infections were reactive with the Enzygnost^® Anti-HIV 1/2 Plus and 12/35 (34%) with the Vironostika^® HIV Ag/Ab. 16 (46%) specimens were confirmed as acute by the INNOTEST^® HIV antigen mAb Antigen test. Only three (9%), 10 (29%) and 9 (27%) specimens were reactive with the Determine HIV-1/2 Ag/Ab Combo, SD Bioline HIV Ag/Ab Combo test and the HIV Combo test, respectively. The specificity of the different tests were 100%, 95%, 100%, 93%, 100% and 93% for Enzygnost^® Anti-HIV 1/2 Plus, Vironostika^® HIV Ag/Ab, INNO-Test HIV antigen mAb, Determine HIV-1/2 Ag/Ab Combo, SD Bioline HIV Ag/Ab Combo test and HIV Combo test respectively.ConclusionRNA test, 4th generation ELISA and Single Ag test are the most sensitive tests for detection of an acute infection. As an alternative, the HIV Combo test is generally slightly more sensitive compared to its previous version, but the SD Bioline HIV Ag/Ab Combo tests has the best performance compared to the other simple rapid tests (SRTs) but none of them are precise in detecting Ag in the determination of acute infections.     

Evaluation of Xpert HIV-1 Qual assay for resolution of HIV-1 infection in samples with negative or indeterminate Geenius HIV-1/2 results

BackgroundDiagnosis of HIV infection is a multistage algorithm. Following screening with 4^th generation combination immunoassay, confirmation of HIV infection is performed with an antibody assay that differentiates HIV-1 from HIV-2 infection. In the newly updated algorithm, samples that are nonreactive or indeterminate in the differentiation assay are to be tested with an HIV-1 nucleic acid amplification (NAAT) test for resolution. Xpert HIV-1 Qual is a new NAAT assay approved for the identification of HIV infection in whole and dried blood.ObjectivesTo assess the performance of Xpert HIV-1 Qual supplementary assay in resolving the clinical status of serum samples reactive by 4^th generation immunoassays and indeterminate or negative by Geenius HIV-1/2 confirmatory assay.Study designIn a retrospective study, samples from 97 individuals for whom the true HIV-1 status was already known (by follow-up samples) and which were negative or indeterminate by HIV-1/2 Geenius assay were tested with Xpert Qual HIV-1 assay.ResultsXpert Qual assay correctly classified all 97 samples from HIV-1 positive (n = 49) and negative (n = 48) individuals. The sensitivity and specificity of Xpert Qual when using the true HIV status as a reference were 100% (92.7–100% at 95% confidence interval [CI] and 92.6–100% at 95% CI, respectively).ConclusionsApplying Xpert Qual HIV-1 assay in the new HIV multi-stage diagnostic algorithm correctly classified 100% of HIV-1 infections including 49 from HIV-1 carriers who have not yet seroconverted. With this assay the total time required for acute HIV diagnosis could be significantly reduced.     

A simple semi-rapid HIV-1&2 confirmatory immunoassay using magnetic particles

The Bionor HIV-1&2 Confirmatory Test is a semi-rapid simple immunoassay based on magnetic particles for the confirmation of serological status to human immunodeficiency virus (HIV). The specificity and sensitivity of this assay was evaluated by comparison with the Diagnostic Biotechnology HIV-1 Western blot (WB) 2.2 and the HIV-2/SBL-6669 WB. Bionor's confirmatory test demonstrated 98% specificity when testing sero-negative blood donors and false positive sera in screening tests compared to 81.5 and 71.6%, respectively, using the HIV-1 WB. The sensitivity of this assay for HIV-1 antibody positive sera was 97.9% compared to the WB which was 99.5%. When testing confirmed HIV-2 antibody positive samples, 2/100 scored negative using this confirmatory test similar to other HIV-2 peptide-based line immunoassays available commercially, whilst 8/100 were indeterminate reacting to HIV-2 membrane antigens only. Bionor's confirmatory test detected HIV-1 seropositivity earlier than the WB in longitudinal seroconversion panels and could discriminate between HIV-1 and -2 infection. The number of indeterminate responses was generally reduced significantly using Bionor's confirmatory test compared to the HIV-1 WB. The greater specificity, speed and ease of interpretation of Bionor's confirmatory test renders it an attractive and cost effective alternative to the WB for confirming HIV serological status worldwide.     

Evaluation of an indirect immunofluorescence assay for confirmation of human immunodeficiency virus type 1 antibody in U.S. blood donor sera.

An indirect immunofluorescence assay (IFA) was evaluated as a confirmatory test for antibody to human immunodeficiency virus type 1 in U.S. blood donor sera previously found to be repeatedly reactive by enzyme immunoassay. IFA results were 100% concordant with a licensed Western blot (immunoblot) for 53 negative and 49 positive samples. Four samples which exhibited antibody to viral proteins from more than one gene, yet were indeterminate by Western blot by the manufacturer's criteria, were also reactive by IFA, whereas 49 additional indeterminate samples were IFA negative.     

Advantages of a human immunodeficiency virus type 1 (HIV-1) persistently infected HeLa T4+ cell line for HIV-1 indirect immunofluorescence serology.

A HeLa T4+ cell line persistently infected with human immunodeficiency virus type 1 (HIV-1) was used in an indirect immunofluorescent antibody assay (IFA) system to explore its potential suitability as an alternative source of viral antigen for confirmatory IFA in HIV serology. In a study of 121 serum samples chosen because they were reactive on repeat examination by enzyme immunoassay but nonspecific by IFA by using HIV-1-infected H9 cells (H9 IFA) or gave discrepant results by enzyme immunoassay and H9 IFA, the specificity and sensitivity of the HeLa T4+ IFA were comparable to those of Western blot (immunoblot), and identification of the true positive samples among these discrepant or nonspecific samples by HeLa T4+ IFA was approximately twice that by H9 IFA. The primary advantages of using the HeLa cell line rather than lymphoid cell lines in IFA are that cells can be grown as a monolayer and that the individual cells are much larger. The cell membrane, cytoplasm, and nucleus are easily discernible; this allows specific and nonspecific staining to be distinguished. At least eight different nonspecific nuclear and cytoplasmic staining patterns were identified in this study by using T4+ cells.     

Multicenter evaluation of a new rapid automated human immunodeficiency virus antigen detection assay

Although human immunodeficiency virus (HIV) antigen assays are of limited value for monitoring antiretroviral therapy, they play an important role for confirmatory testing of fourth generation HIV screening enzyme immunoassay (EIA) reactive samples. In a multicenter study, a new automated rapid p24 antigen assay, Elecsys HIV Ag (Roche Diagnostics Boehringer Mannheim GmbH, Penzberg, Germany), was compared to FDA licensed tests (Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay). In the evaluation 27 seroconversion panels were included, sera from the acute phase of infection, single and follow-up samples from HIV antibody positive patients, dilution series of HIV antigen positive standards, sera and cell culture supernatants infected with different HIV-1 subtypes (A-H, and O) HIV-2 and recombinant HIV-1 (gag/env) isolates. To challenge the specificity of the new assay, 2565 unselected blood donors, sera from pregnant women, dialysis and hospitalized patients and 407 potentially cross-reactive samples were investigated. Acute HIV infection was detected in three to eight seroconversion panels earlier with Elecsys HIV Ag than with the alternative assays. Higher numbers of serum samples from HIV infected patients tested positive by Elecsys HIV Ag than with the comparative assays. All HIV-1 subtypes and HIV-2 isolates were recognized with Elecsys HIV Ag. Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay showed a variable sensitivity for the different HIV-1 subtypes. The specificity of Elecsys HIV Ag and Coulter HIV-1 p24 antigen assay were 99.8 and 99.93%, respectively. All the eight sera that were false reactive by Elecsys HIV Ag were tested negative with the Elecsys HIV Ag Neutralization Test. In conclusion, Elecsys HIV Ag was more sensitive than the alternative assays and showed a high specificity in combination with the neutralization assay. The very short incubation time of 18 min and the fully automated procedure of Elecsys HIV Ag which permits direct testing from the primary patient blood collection tube, represent a major improvement for routine laboratory diagnosis in comparison to the alternative assays.     

HIV-1 antibody testing strategy: evaluation of ELISA screening and western blot profiles in a mixed low risk/high risk patient population

The Westmead HIV-1 antibody testing strategy showed that, regardless of ELISA screening kit manufacturer, sera which were repeatedly positive by two ELISA screening assays (one indirect and the other competitive format) had a 97-98% chance of being confirmed positive by Western blot for HIV-1 antibody or a less than 3% chance of either being identified as a seroconverter (1%) or a late stage AIDS patient (1.2%). Sera which were discordant by two ELISA screening assays had a less than 4% chance of either being confirmed positive by Western blot (2.5%) or identified as a seroconverter (1.3%). The incidence of non-specific indeterminate Western blot profiles were shown to be inversely proportional to the specificity of the ELISA screening kits used. The use of a recombinant envelope ELISA was able to confirm the viral specificity of HIV-1 envelope bands (gp160, 120 or 41) on Western blot. Guidelines suggested by the Australian National HIV Reference Laboratory, Fairfield Hospital, Melbourne, which categorized indeterminate or typical Western blot profiles into four reaction groups were found to be useful for the interpretation of Western blot patterns. A Western blot profile which is reactive for HIV-1 viral glycoproteins (gp160, 120 and 41) alone or in combination with not more than two other viral proteins (Indeterminate Group 4) and which is confirmed viral envelope specific by a recombinant envelope ELISA can be used as a predictor of seroconversion.     

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.     

Performance of Determine Combo and other point-of-care HIV tests among Seattle MSM

Background and objectiveThe rapid test study was a real-time comparison of point-of-care (POC) HIV tests to determine their abilities to detect early HIV infection.Study designMen and transgender persons reporting sex with men in the prior year were recruited at the Public Health—Seattle & King County STD Clinic, Gay City Health Project, and University of Washington Primary Infection Clinic. Study tests included the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test performed on oral fluids and tests performed on fingerstick whole blood specimens including OraQuick, Uni-Gold Recombigen HIV test, Determine HIV-1/2 Ag/Ab Combo, and INSTI HIV-1 Rapid Antibody Test. Specimens from subjects with negative results were sent for EIA and nucleic acid amplification testing. McNemar's exact tests compared the numbers of HIV-infected subjects detected.ResultsBetween February 2010 and August 2014, there were 3438 study visits. Twenty-four subjects had discordant POC results with at least one reactive and one non-reactive test, including one subject with a reactive Determine p24 antigen. OraQuick performed on oral fluids identified fewer persons compared to all fingerstick tests. OraQuick performed on fingerstick whole blood detected fewer persons compared to the Determine Combo antibody component (p = .008) and Combo overall (p = .004), and there was a trend when compared to INSTI (p = .06). The Determine Combo specificity was 98.99%.ConclusionsAs reported by others, Determine Combo underperforms compared to laboratory-based testing, but it did detect one acute infection. If these results are validated, the specificity of Determine Combo may limit its usefulness in populations with lower HIV incidence.     

Comparison of the clinical performance of the ECLusys HIV combi assay with the Lumipulse f and HISCL 2000-i HIV-1/2 ab screening assays

We compared the ECLusys HIV combi assay (ECL HIV Ag/Ab) to the Lumipulse Forte (LPf HIV 1/2 Ab) and HISCL (HIS HIV 1/2 Ab) assays. In a dilution sensitivity test using dilution panels of WHO HIV antibody international reference panel (HIV-1 Subtype A, B, C, E, HIV-1 Group O, HIV-2) and HIV-1/2 Ab CE marked material(HIV-1, HIV-2) parent specimens, the ECL assay enabled detection at a higher level of sensitivity than either the LPf assay or the HIS assay for all dilution panels. In an early detection test in the early phase of infection in which a BBI HIV seroconversion panel was used, the ECL assay enabled detection 7 days after initial blood sample collection, whereas the LPf and HIS assays enabled detection after 27 days. In a specificity test using high RF positive specimens (n=33), pregnancy specimens (n=35), cytomegalovirus antibody positive specimens (n=36), and high M protein positive specimens (n=21) that were confirmed negative for HIV-1/2 antibodies by the LPf assay, negative results were obtained for all specimens on both the ECL assay and the HIS assay. In a correlation test using routinely collected clinical specimens (n=121), including positive stock specimens, the ECL and HIS assays demonstrated the highest agreement rate 98.3%. The above results confirmed that the fourth-generation reagent ECL assay, which simultaneously detects both HIV-1/2 antibodies and p24 antigens, is both highly sensitive and specific, and is a suitable assay for use in routine testing.