HIV Diagnostic Algorithm Research Articles

HIV Diagnostic Algorithms in A Resource-Limited Setting: A Comparative Study

Background: Adoption of appropriate HIV testing algorithm is essential for quality HIV diagnostic results. Whether rapid enzyme immunoassay (rEIA) or combination of rapid enzyme immunoassay and enzyme-linked immunosorbent assay (rEIA-ELISA) alternative confirmatory algorithm is enough to make accurate HIV diagnosis has been a subject of controversies. Recent evaluation of national algorithm for HIV testing presented some discrepancies in results. Again, current guideline prescribed discontinuity of Western blot supplemental test and its replacement with Geenius HIV-1/ HIV-2 differentiation test followed by nucleic acid testing. The objective of this study is to compare HIV-1 testing algorithms using ELISA-NAT algorithm as the gold standard for HIV-1 diagnosis, and rEIA-Western blot and ELISA-Western blot as alternative confirmatory algorithms for HIV-2 diagnosis. Methods: This is a comparative study involving a cohort of 173 (98.9%) HIV-positive subjects and two (1.1%) HIV-negative subjects initially diagnosed on the basis of rEIA algorithm. Plasma samples separated from ethylene diamine tetra-acetic acid anticoagulated blood were used for further analyses. Fourth generation HIV antigen-antibody kit was used for further testing to make up rEIA-ELISA algorithm. Analyses with Western blot 1 and 2 assays were done as supplemental tests to make up alternative rEIA-Western blot and ELISA-Western blot confirmatory algorithms. Research subjects were further tested for HIV-RNA under PEPFAR plan. The sensitivity, specificity, positive predictive value and negative predictive value of algorithms were compared using the descriptive statistics of the SPSS version 17. Results: The HIV testing analyses showed 100% sensitivity, specificity, positive predictive value and negative predictive value from algorithm to algorithm especially for HIV-1 diagnosis. Use of combined ELISA-NAT confirmatory algorithm validated outcomes of rEIA-Western blot and ELISA-Western blot alternative confirmatory algorithms for HIV-1 diagnosis. Among HIV-1 positive samples tested, 50.0-60.7% HIV-2 Western blot indeterminate result was obtained and 7.1-10.7% HIV-1/HIV-2 co-infections were observed. Conclusion: The rEIA or national algorithm and its combination with enzyme-linked immunosorbent assay are reliable alternative confirmatory algorithms for the diagnosis of established HIV-1infection in resource-limited settings but not for HIV-2. Diagnosing acute HIV infection with current algorithm is limited and still require further review.

Implementation of an HIV RNA Qualitative PCR Assay in an HIV Diagnostic Algorithm: A Single-Institution Experience.

Boston Medical Center (BMC) is a private, not-for-profit 514-bed academic medical center and legacy safety net hospital serving a diverse global patient population. BMC recently implemented a new HIV-1/HIV-2 Qualitative RNA PCR (HIV RNA QUAL) cleared by the US Food and Drug Administration to (1) replace antibody discrimination follow-up testing after a reactive fourth-generation (4G) serology screen and (2) use as a stand-alone diagnostic for suspected seronegative acute HIV infection. This report summarizes the results of a production monitor for the first 3 months postimplementation. The monitor characterized test utilization, diagnostic turnaround time, impact on send-out testing, results reflexed to HIV RNA discrimination follow-up, and discrepancies between screening and HIV RNA results that necessitated additional investigation. Another element was the novelty of using HIV RNA QUAL while awaiting the existing Centers for Disease Control and Prevention HIV testing algorithm update. The 4G screening components and the HIV RNA QUAL were also used to create an algorithm specific to and compliant with current guidelines for screening patients on HIV preexposure prophylaxis. Based on our findings, this new test algorithm may be reproducible and instructive at other institutions.

Real-world HIV diagnostic testing patterns in the United States.

To understand real-world implementation of the updated CDC HIV diagnostic testing algorithm. Retrospective database analysis. Using data from Quest Diagnostics, we identified patients with at least 1 HIV-1/HIV-2 antibody differentiation test (BioRad Geenius HIV 1/2 Supplemental Assay [Geenius]) between January 1 and December 31, 2017. Study measures included Health Insurance Portability and Accountability Act-compliant patient demographics, test results, test frequency, and sequence relative to the CDC HIV diagnostic algorithm, including HIV-1 RNA Qualitative Assay (Aptima) or HIV-2 nucleic acid test (NAT). A total of 26,319 patients were identified (mean [SD] age, 40.7 [14.3] years; 66.4% male), with 28,954 Geenius tests, 7234 Aptima tests, and 298 HIV-2 NATs. In 26.4% of test sequences, the Geenius results were indeterminate or negative and required subsequent confirmatory NATs. A total of 8.5% of patients had more than 1 Geenius test in 2017, and 11.2% of the time, results of the first and second tests differed. A total of 74.2% of test sequences matched the CDC-recommended algorithm. Our study findings suggest that the CDC HIV diagnostic algorithm is complex and may pose suboptimal testing efficiency. Opportunities to improve diagnostic efficiency by reducing indeterminate results and repeat tests are warranted.

Early Diagnosis of HIV-1 and HIV-2 Using Cobas HIV-1/HIV-2 Qualitative Test: A Novel Qualitative Nucleic Acid Amplification Test for Plasma, Serum, and Dried Blood Spot Specimens.

Background:Nucleic acid amplification tests (NATs) minimize the time from HIV infection to diagnosis, reducing transmission during acute HIV. NATs are especially useful for diagnosing HIV in children younger than 18 months and discriminating between HIV-1 and HIV-2.Methods:We evaluated the performance of the cobas HIV-1/HIV-2 qualitative (cobas HIV-1/2 Qual) test for use on cobas 6800/8800 Systems. The results of adult plasma and serum samples and pediatric dried blood spots were compared with those of the recomLine HIV-1 & HIV-2 Immunoglobulin G serological test and COBAS AmpliPrep/COBAS TaqMan HIV-1 qualitative test, v2.0. Genotype inclusivity and limits of detection were determined, and sensitivity on seroconversion panels was compared with that in the Bio-Rad Geenius HIV 1/2 Confirmatory Assay, Abbott ARCHITECT HIV Ag/Ab Combo serological test, and cobas TaqScreen MPX, v2.0.Results:Concordance of cobas HIV-1/2 Qual test with the comparator serological test and COBAS AmpliPrep/COBAS TaqMan test was ≥99.6% with all sample types. Reactivity with all HIV genotypes was 100%. LOD in plasma samples was 14.8, 12.6, and 27.9 copies/mL for HIV-1 group M, HIV-1 group O, and HIV-2, respectively, with similar results for serum samples. LOD in dried blood spots was 255 copies/mL for HIV-1 and 984 copies/mL for HIV-2. HIV infection was detected 18.9 days and 8.5 days earlier than the confirmatory and serological assays, respectively, and at a similar time to the NAT.Conclusions:The cobas HIV-1/2 Qual test enables early and accurate diagnoses of HIV-1 and HIV-2 in adults and children across sample types. The assay could help avert transmission during acute HIV, simplify HIV diagnostic algorithms, and promote the survival of HIV-infected children.

Verification and Implementation of HIV Antibody Differentiation Testing to Improve Turnaround Time for the HIV Diagnostic Algorithm.

Relying on reference laboratories for HIV confirmation testing may lead to delays in treatment and can cause stress for patients who have positive HIV screening results. To internalize HIV-1/HIV-2 antibody differentiation testing within the hospital laboratory. We analytically verified an HIV antibody differentiation immunoassay and subsequently compared result turnaround times (TATs) for HIV antibody differentiation and HIV-1 qualitative RNA in the months before and after the test internalization. HIV antibody differentiation was successfully verified. TATs for HIV antibody differentiation and HIV-1 RNA significantly improved, from medians of 40.4 hours and 156.5 hours to medians of 17.7 hours and 56.5 hours, respectively, after the internalization. The 90th-percentile turnaround times declined by 72% and 44%, respectively. It is feasible for a hospital laboratory to verify HIV antibody-differentiation testing. Its implementation may considerably improve result TATs for the HIV diagnostic algorithm.

HIV diagnostic algorithm requires confirmatory testing for initial indeterminate or positive screens in the first week of life.

Risk for nondiagnostic and false-positive HIV testing has not been quantified for neonates. From April 2015 to July 2018, we screened HIV-exposed infants in Botswana less than 96 h from birth by qualitative DNA PCR. Repeat blood draws for DNA and RNA PCR testing occurred for initial positive and indeterminate results to establish final diagnosis. We compared screening DNA PCR cycle threshold values with final HIV status of the child. Of 10 622 HIV-exposed infants, 10 549 (99.3%) had no HIV DNA detected (negative), 42 (0.4%) had HIV DNA detected (positive), and 31 (0.3%) tested indeterminate at first HIV screen. Repeat testing identified 2 (5.0%) of 40 positive screens (2 declined additional testing) as false positives and confirmed 2 (6.5%) of 31 indeterminate screens as infected. Median cycle threshold value at screening was 28.1 (IQR 19.8--34.8) for children with final positive status, and 35.5 (IQR 32.8--41.4) for indeterminates who were ultimately negative. Six (15%) of 40 infants with final positive status had cycle threshold value greater than 33 at first screen, whereas 3 (9.7%) of 31 indeterminates with final negative status had cycle threshold value 33 or less at first screen. This threshold resulted in a negative predictive value of 82% and a positive predictive value of 92% for a single screen. Although a DNA PCR cycle threshold value of 33 was predictive of the final HIV status in newborns, overlap occurred for true positives, false positives, and initial indeterminates. Testing additional samples should be standard practice for positive and indeterminate HIV DNA PCR tests in the first week of life.

Could HIV-1 RNA Testing be an Option as the Second Step in the HIV Diagnostic Algorithm?

There is benefit to early HIV-1 diagnosis and treatment, but there is no Food and Drug Administration-approved quantitative assay with a diagnostic claim. We compared the performance of the Hologic Aptima HIV-1 Quant (APT-Quant) and Aptima HIV-1 Qual (APT-Qual) assays for diagnostic use and the performance of a diagnostic algorithm consisting of Bio-Rad BioPlex 2200 HIV Ag-Ab assay (BPC) followed by APT-Quant (2-test) compared with BPC followed by Geenius HIV-1/2 supplemental assay (Geenius) with reflex to APT-Qual (3-test). Five hundred twenty-four plasma, which included 419 longitudinal specimens from HIV-1 seroconverters (78 were after initiating antiretroviral therapy [ART]) and 105 from ART-naive persons with established HIV-1 infections, were used to evaluate APT-Quant performance for diagnostic use. Specimens from 200 HIV-negative persons were used to measure specificity. For the algorithm comparison, BPC-reactive specimens were evaluated with the 2-test or 3-test algorithm. McNemar's test was used to compare performance. The APT-Quant detected more samples early in infection compared with APT-Qual. The APT-Quant specificity was 99.8%. Before ART initiation, the algorithms performed similarly among samples from different stages of infection. After ART initiation, the 3-test algorithm performed significantly better (P = 0.0233). The APT-Quant has excellent performance for diagnostic use. The 2-test algorithm works well in ART-naive samples, but its performance decreases after the IgG response is elicited and with ART-induced suppressed viremia. Providing confirmation and viral load assay with 1 test result could be advantageous for patient care. However, additional factors and challenges associated with the implementation of this 2-test algorithm, such as cost, specimen type, and collection need further evaluation.

Performance of an Alternative Laboratory-Based HIV Diagnostic Testing Algorithm Using HIV-1 RNA Viral Load.

Since 2014, the recommended algorithm for laboratory diagnosis of HIV infection in the United States has consisted of an HIV-1/2 antigen/antibody (Ag/Ab) test followed by an HIV-1/2 antibody (Ab) differentiation test and, if necessary, a diagnostic HIV-1 nucleic acid test to resolve discordant or indeterminate results. Using stored specimens from persons seeking HIV testing who had not received a previous diagnosis or treatment, we compared the performance of a 3-step alternative algorithm consisting of an Ag/Ab test followed by a quantitative HIV-1 RNA viral load assay and, if viral load is not detected, an Ab differentiation test, to that of the recommended algorithm. We calculated the sensitivity and specificity of 5 Ag/Ab tests and the proportion of specimens correctly classified by the alternative algorithm compared with the recommended algorithm. Results were examined separately for specimens classified as early infection, established infection, and false-reactive screening. Sensitivity and specificity were similar among all Ag/Ab tests. Viral load quantification correctly classified all specimens from early infection, all false-reactive screening specimens, and the majority of specimens from established infection. Although cost, regulatory barriers, test availability, and the ability to differentiate early from established infection must be considered, this alternative algorithm can potentially decrease the total number of tests performed and reduce turnaround time, thereby streamlining HIV diagnosis and initiation of treatment.

Rapid Testing Algorithm Performance in a Low-Prevalence Environment.

The performance of a statewide HIV rapid test algorithm (RTA) in a low-prevalence setting (0.71%) was examined for 3 years. An initial rapid screening by HIV-1/2 Ag/Ab Combo test (RT#1) with Ab verification using a second, different rapid test (RT#2) was conducted. Clinic referral was immediate for antigen-only-positive screens. Antibody-positive screens were confirmed by RT#2. Specimens were collected following discordant RTA results (initially Ab-POS by RT#1, but negative on RT#2) and tested in accordance with the current Centers for Disease Control and Prevention/Association of Public Health Laboratories-based HIV diagnostic algorithm supplemented by a quantitative viral load whenever possible. Of 310,785 tests performed, 2400 preliminary positive screens were identified; 2191 (91.8%) confirmed by RT#2. Of 13 Determine Combo AG-POS results identified, only 1 confirmed positive. Of the remaining 196 discordant results, 182 (92.9%) were uninfected, including 13 with AG-POS/AB-POS results. Of 14 true positives (7.1%) identified after discordant RTA results, the average quantitative HIV-1 viral load was 277,385 copies/mL, but 5 (35.7%) of 14 had viral loads <1000 copies/mL. Among the 2191 "presumptive positive" by RTA, 3 false-positive (FP) RTAs were reported (both rapid tests having positive results, while the HIV-1/2 Ag/Ab assay and quantitative HIV-1 viral load showed negative results). The RTA was effective in predicting true-positive HIV test results and facilitating linkage to care. Discordant results were infrequent. Fingerstick DC Ag detection identified a single early infection. Many discordant cases that were subsequently positive were associated with viral loads <1000 copies/mL.

1782. Real-World HIV Diagnostic Testing Patterns in the United States

BackgroundCurrent HIV diagnostic laboratory testing guidelines from the US Centers for Disease Control and Prevention (CDC) recommend a sequence of tests for detection, differentiation, and confirmation of HIV-1 and HIV-2 diagnosis. There is a gap in knowledge about real-world implementation of the testing algorithm. The aim of this study was to characterize the population that underwent HIV antibody differentiation and confirmatory testing and to describe subsequent testing patterns from a large US clinical laboratory database.MethodsPatients who received one or more HIV-1/2 antibody differentiation test (BioRad Geenius™ HIV 1/2 Supplemental Assay [Geenius]) in the Quest Diagnostics laboratory database between January 1, 2017 and December 31, 2017 were selected into the study; earliest test date was index date. Geenius tests, HIV-1 qualitative RNA (Aptima HIV-1 RNA Qualitative Assay [Aptima]), and HIV-2 DNA/RNA confirmatory tests subsequent to index date were captured. Study measures included pt demographic characteristics, testing frequency and sequencing, and test results. For patients with >1 Geenius test in 2017, concordance between index and subsequent test results was assessed.ResultsThere were 26,319 unique patients identified who received ≥1 HIV antibody differentiation result from the Geenius assay. Mean age was 40.7 ± 14.3 years, 66.4% were male, and 42.5% were from southern states. Among the study population, there were 28,954 Geenius, 7,234 Aptima, and 298 HIV-2 DNA/RNA confirmatory tests. 26.4% of Geenius test results were discordant with the initial positive fourth-generation HIV screening results and required subsequent confirmatory testing. In terms of sequencing, the CDC-recommended HIV diagnostic algorithm was followed 74% of the time after screening. 8.5% of patients had >1 Geenius test in 2017; 11.2% of the retests returned different results compared with the first test.ConclusionThe CDC recommended algorithm for HIV diagnosis is complex for laboratories to implement and currently available assays do not support testing efficiency. To mitigate observed inefficiencies and reduce the laboratory burden of HIV testing, a more accurate and reliable approach for HIV differentiation and confirmatory testing is needed.Disclosures All authors: No reported disclosures.

Performance evaluation of the Bio-Rad Geenius HIV 1/2 supplemental assay

BackgroundIn the US, the HIV diagnostic algorithm for laboratory settings recommends the use of an HIV-1/HIV-2 differentiation supplemental assay after an initial reactive antigen/antibody (Ag/Ab) assay result. Since the discontinuation of the Multispot HIV-1/HIV-2 Rapid Test (MS), the Geenius HIV-1/2 Supplemental assay (Geenius) is the only FDA-approved supplemental differentiation test. ObjectiveWe compared the performance of Geenius to MS and Western Blot (WB). Study designThe relative seroconversion plasma reactivity of Geenius and MS was assessed using a 50% cumulative frequency analysis from 17 HIV-1 seroconverters. In addition, previously characterized plasma specimens, 186 HIV-1 positive, 100 HIV-2 positive, and 93 Ag/Ab-positive/HIV-1 RNA-negative, were tested with Geenius v1.1 software. McNemar’s test was used for paired comparison analysis. A subset of 48 specimens were retested with the upgraded Geenius v1.3 software. ResultsIn HIV-1 seroconverters, the relative seroconversion reactivity was 2.5 and 2 days before the first positive HIV-1 WB for Geenius and MS, respectively. In HIV-1 positive samples, Geenius performed similarly to HIV-1 WB (p=0.1687) and MS (p=0.8312). In HIV-2 positive samples, Geenius underperformed compared to HIV-2 WB (p=0.0005) and MS (p=0.0012). When using the upgraded software among the HIV-1 positive and Ag/Ab-reactive/HIV-1 RNA-negative samples, gp140 reactivity decreased without affecting characterization of HIV-2 samples. ConclusionsWith HIV-1 samples, Geenius, WB and MS performance was similar as supplemental tests. The updated Geenius software reduced false gp140 reactivity, but had no impact on identifying true HIV-2 infections. Further evaluation will assess the impact of the Geenius software update on final diagnostic interpretations

J-104 Geenius HIV 1/2 Supplemental Assay: A Rapid, Reliable and Simple System for the Confirmation and Differentiation of Antibodies to HIV

INTRODUCTION: In 2017 Bio-Rad Laboratories completed launching the FDA-approved Geenius HIV-1/HIV-2 Supplemental assay to replace Multispot HIV-1/HIV-2 in the current CDC HIV Diagnostic Algorithm. The Geenius cassette contains antibody-binding protein A, which is conjugated to colloidal gold dye particles; HIV-1 (p31, gp160, p24, gp41) and HIV-2 (gp36, gp140) antigens are bound to the membrane solid phase. Serum, plasma, or whole blood may be tested. Geenius uses an automated cassette reader and proprietary software to interpret HIV-1 and HIV-2 results. We report more recent performance data for this system, as well as an investigational protocol for testing dried blood spot (DBS) specimens. METHODS: (1) WHO evaluated the Geenius HIV-1/HIV-2 Supplemental testing system with a panel of 1117 specimens. (2) DBS specimens were tested on Geenius using an investigational procedure (40 μL of DBS eluate and one drop of assay buffer). Samples included established infections (131 HIV-1, 31 HIV-2, one dual), 60 seroconversion specimens, and 106 DBS collected by CDC during HIV surveillance. RESULTS: (1) Geenius sensitivity in the WHO evaluation was 100% and specificity was 97.4%. Seroconversion samples were detected +0.875 days vs. the benchmark. All the mixed titer and reference specimens were correctly classified. (2) Geenius DBS correctly detected HIV-1 and HIV-2 DBS with minor exceptions. DBS seroconversions showed slightly reduced sensitivity vs. plasma. CONCLUSIONS: (1) The Geenius HIV-1/2 Confirmatory Assay was accepted for the WHO list of prequalified diagnostics (March 17, 2017). (2) Results of an investigational Geenius DBS procedure appear promising for settings without capacity for venipuncture.

Trends in testing algorithms used to diagnose HIV infection, 2011–2015, United States and 6 dependent areas

BackgroundIn 2014 the Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories (APHL) issued updated laboratory testing recommendations for the diagnosis of HIV infection. ObjectivesTo examine trends in the use of HIV diagnostic testing algorithms, and determine whether the use of different algorithms is associated with selected patient characteristics and linkage to HIV medical care. Study designAnalysis of HIV infection diagnoses during 2011–2015 reported to the National HIV Surveillance System through December 2016. Algorithm classification: traditional = initial HIV antibody immunoassay followed by a Western blot or immunofluorescence antibody test; recommended = initial HIV antibody IA followed by HIV-1/2 type-differentiating antibody test; rapid = two CLIA-waived rapid tests on same date. ResultsDuring 2011–2015, the percentage of HIV diagnoses made using the traditional algorithm decreased from 84% to 16%, the percentage using the recommended algorithm increased from 0.1% to 64%, and the percentage using the rapid testing algorithm increased from 0.1% to 2%. The percentage of persons linked to care within 30 days after HIV diagnosis in 2015 was higher for diagnoses using the recommended algorithm (59%) than for diagnoses using the traditional algorithm (55%) (p < 0.05). ConclusionsDuring 2011–2015, the percentage of HIV diagnoses reported using the recommended and rapid testing algorithms increased while the use of the traditional algorithm decreased. In 2015, persons with HIV diagnosed using the recommended algorithm were more promptly linked to care than those with diagnosis using the traditional algorithm.

The Importance of 4th Generation HIV Testing in an Urban Emergency Department

Early Acute Human Immunodeficiency Virus Infection (eAHI) diagnosis, via 4th generation testing methodology, presents an opportunity for earlier detection and immediate linkage to care for infected persons. We report on two patients with high-risk behaviors for HIV infection, presenting with atypical symptoms of eAHI in an urban Emergency Department (ED). This case report should raise the index of suspicion for HIV among ED physicians as well as underscore the importance of reducing HIV transmission through earlier detection. Universal screening of patients aged 13–64, incorporating new HIV diagnostic algorithms, is recommended by the Centers for Disease Control and Prevention (CDC). By employing the 4th generation HIV testing methodology, we can potentially diagnose HIV infection earlier compared to older testing methodologies. Currently, 3rd generation HIV testing is used to detect the presence of HIV antibodies, generally through an enzyme-linked immunosorbent assay (ELISA). However, detection of HIV antibodies can take anywhere from 3 to 12 weeks, depending on the individual and testing modality used. This newer diagnostic paradigm enables earlier identification of newly infected individuals. Early HIV detection allows for linkage to care and the administration of effective treatment modalities shortly thereafter. As HIV transmission is highest during its initial acquisition, early detection and linkage to care has been shown to be an efficient method to decrease transmission through subsequent changes in behaviors of those infected.

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.

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay

ObjectiveFDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm. MethodsBRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms. ResultsBRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p=0.0133), but fewer infections than BRC-plasma (p=0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p=0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens. ConclusionsThe DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture.

Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm

BackgroundThe 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients’ blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. ObjectivesTo validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2–infected Senegalese study participants. Study designWe evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. ResultsAll PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. ConclusionsOur TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm.

Accounting for False Positive HIV Tests: Is Visceral Leishmaniasis Responsible?

BackgroundCo-infection with HIV and visceral leishmaniasis is an important consideration in treatment of either disease in endemic areas. Diagnosis of HIV in resource-limited settings relies on rapid diagnostic tests used together in an algorithm. A limitation of the HIV diagnostic algorithm is that it is vulnerable to falsely positive reactions due to cross reactivity. It has been postulated that visceral leishmaniasis (VL) infection can increase this risk of false positive HIV results. This cross sectional study compared the risk of false positive HIV results in VL patients with non-VL individuals.Methodology/Principal FindingsParticipants were recruited from 2 sites in Ethiopia. The Ethiopian algorithm of a tiebreaker using 3 rapid diagnostic tests (RDTs) was used to test for HIV. The gold standard test was the Western Blot, with indeterminate results resolved by PCR testing. Every RDT screen positive individual was included for testing with the gold standard along with 10% of all negatives. The final analysis included 89 VL and 405 non-VL patients. HIV prevalence was found to be 12.8% (47/ 367) in the VL group compared to 7.9% (200/2526) in the non-VL group. The RDT algorithm in the VL group yielded 47 positives, 4 false positives, and 38 negatives. The same algorithm for those without VL had 200 positives, 14 false positives, and 191 negatives. Specificity and positive predictive value for the group with VL was less than the non-VL group; however, the difference was not found to be significant (p = 0.52 and p = 0.76, respectively).ConclusionThe test algorithm yielded a high number of HIV false positive results. However, we were unable to demonstrate a significant difference between groups with and without VL disease. This suggests that the presence of endemic visceral leishmaniasis alone cannot account for the high number of false positive HIV results in our study.

Potential for false-positive HIV test results using rapid HIV testing algorithms.

In order to scale up access to HIV counselling and testing in Nigeria, an HIV diagnostic algorithm based on rapid testing was adopted. However, there was the need to further evaluate the testing strategy in order to better assess its performance, because of the potential for false positivity. The objective of this study was to compare positive HIV test results obtained from the approved rapid testing algorithm with results from western blot tests performed on samples from the same patient. A retrospective review was conducted of HIV screening and confirmatory results for patients seen between 2007 and 2008. Rapid test and western blot results were extracted and compared for concordance. Discordant results were further reviewed using a combination of HIV-1 RNA viral load and CD4+ cell count test results and clinical presentation from medical records. Analysis of 2228 western blot results showed that 98.3% (n = 2191) were positive for HIV-1, 0.4% (n = 8) were positive for HIV-2 and 0.3% (n = 7) were dual infections (positive for both HIV-1 and HIV-2); 0.6% (n = 13) were indeterminate and 0.4% (n = 9) were negative. Further investigation of the 13 indeterminate results showed nine to be HIV-1 positive and four to be HIV-negative, for a total of 13 negative results. The positive predictive value of the HIV counselling and testing algorithm was 99.4%. Using the rapid testing algorithm alone, false positives were detected. Therefore, effective measures such as training and retraining of staff should be prioritised in order to minimise false-positive diagnoses and the associated potential for long-term psychological and financial impact on the patients.

Nucleic acid testing by public health referral laboratories for public health laboratories using the U.S. HIV diagnostic testing algorithm

BackgroundMany public health laboratories adopting the U.S. HIV laboratory testing algorithm do not have a nucleic acid test (NAT), which is needed when the third- or fourth-generation HIV screening immunoassay is reactive and the antibody-based supplemental test is non-reactive or indeterminate. ObjectivesAmong public health laboratories utilizing public health referral laboratories for NAT conducted as part of the algorithm, we evaluated the percentage of screening immunoassays needing NAT, the number of specimens not meeting APTIMA (NAT) specifications, time to APTIMA result, the proportion of acute infections (i.e., reactive APTIMA) among total infections, and screening immunoassay specificity. Study designFrom August 2012 to April 2013, 22 laboratories enrolled to receive free APTIMA (NAT) at New York or Florida public health referral laboratories. Data were analyzed for testing conducted until June 2013. ResultsSubmitting laboratories conducted a median of 4778 screening immunoassays; 0–1.3% (median 0.2%) needed NAT. Of 140 specimens received, 9 (6.4%) did not meet NAT specifications. The median time from specimen collection to reporting the 11 reactive NAT results was ten days, including six days from receipt in the submitting laboratory to shipment to the referral laboratory. Acute infections ranged from 0 to 12.5% (median 0%) of total infections. Third- and fourth-generation immunoassays met package insert specificity values. ConclusionsPublic health referral laboratories provide a feasible option for conducting NAT. Reducing the time from specimen collection to submission of specimens for NAT is an important step toward maximizing the public health impact of identifying acute infections.