Designing HIV Testing Algorithms Based on 2015 WHO Guidelines Using Data from Six Sites in Sub-Saharan Africa.

Our objective was to evaluate the performance of HIV testing algorithms based on WHO recommendations, using data from specimens collected at six HIV testing and counseling sites in sub-Saharan Africa (Conakry, Guinea; Kitgum and Arua, Uganda; Homa Bay, Kenya; Douala, Cameroon; Baraka, Democratic Republic of Congo). A total of 2,780 samples, including 1,306 HIV-positive samples, were included in the analysis. HIV testing algorithms were designed using Determine as a first test. Second and third rapid diagnostic tests (RDTs) were selected based on site-specific performance, adhering where possible to the WHO-recommended minimum requirements of ≥99% sensitivity and specificity. The threshold for specificity was reduced to 98% or 96% if necessary. We also simulated algorithms consisting of one RDT followed by a simple confirmatory assay. The positive predictive values (PPV) of the simulated algorithms ranged from 75.8% to 100% using strategies recommended for high-prevalence settings, 98.7% to 100% using strategies recommended for low-prevalence settings, and 98.1% to 100% using a rapid test followed by a simple confirmatory assay. Although we were able to design algorithms that met the recommended PPV of ≥99% in five of six sites using the applicable high-prevalence strategy, options were often very limited due to suboptimal performance of individual RDTs and to shared falsely reactive results. These results underscore the impact of the sequence of HIV tests and of shared false-reactivity data on algorithm performance. Where it is not possible to identify tests that meet WHO-recommended specifications, the low-prevalence strategy may be more suitable.

Assessment of desiccants and their instructions for use in rapid diagnostic tests.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are protected from humidity-caused degradation by a desiccant added to the device packaging. The present study assessed malaria RDT products for the availability, type and design of desiccants and their information supplied in the instructions for use (IFU). METHODS: Criteria were based on recommendations of the World Health Organization (WHO), the European Community (CE) and own observations. Silica gel sachets were defined as self-indicating (all beads coated with a humidity indicator that changes colour upon saturation), partial-indicating (part of beads coated) and non-indicating (none of the beads coated). Indicating silica gel sachets were individually assessed for humidity saturation and (in case of partial-indicating silica gels) for the presence of indicating beads. RESULTS: Fifty malaria RDT products from 25 manufacturers were assessed, 14 (28%) products were listed by the "Global Fund Quality Assurance Policy" and 31 (62%) were CE-marked. All but one product contained a desiccant, mostly (47/50, 94%) silica gel. Twenty (40%) RDT products (one with no desiccant and 19 with non-indicating desiccant) did not meet the WHO guidelines recommending indicating desiccant. All RDT products with self- or partial-indicating silica gel (n = 22 and 8 respectively) contained the toxic cobalt dichloride as humidity indicator. Colour change indicating humidity saturation was observed for 8/16 RDT products, at a median incidence of 0.8% (range 0.05%-4.6%) of sachets inspected. In all RDTs with partial-indicating silica gel, sachets with no colour indicating beads were found (median proportion 13.5% (0.6%-17.8%) per product) and additional light was needed to assess the humidity colour. Less than half (14/30, 47%) IFUs of RDT products with indicating desiccants mentioned to check the humidity saturation before using the test. Information on properties, safety hazards and disposal of the desiccant was not included in any of the IFUs. There were no differences between Global Fund-listed and CE marked RDT products compared to those which were not. Similar findings were noted for a panel of 11 HIV RDTs that was assessed with the same checklist as the malaria RDTs. CONCLUSION: RDTs showed shortcomings in desiccant type and information supplied in the IFU.

HIV misdiagnosis in sub-Saharan Africa: performance of diagnostic algorithms at six testing sites.

INTRODUCTION: We evaluated the diagnostic accuracy of HIV testing algorithms at six programmes in five sub-Saharan African countries. METHODS: In this prospective multisite diagnostic evaluation study (Conakry, Guinea; Kitgum, Uganda; Arua, Uganda; Homa Bay, Kenya; Doula, Cameroun and Baraka, Democratic Republic of Congo), samples from clients (greater than equal to five years of age) testing for HIV were collected and compared to a state-of-the-art algorithm from the AIDS reference laboratory at the Institute of Tropical Medicine, Belgium. The reference algorithm consisted of an enzyme-linked immuno-sorbent assay, a line-immunoassay, a single antigen-enzyme immunoassay and a DNA polymerase chain reaction test. RESULTS: Between August 2011 and January 2015, over 14,000 clients were tested for HIV at 6 HIV counselling and testing sites. Of those, 2786 (median age: 30; 38.1% males) were included in the study. Sensitivity of the testing algorithms ranged from 89.5% in Arua to 100% in Douala and Conakry, while specificity ranged from 98.3% in Doula to 100% in Conakry. Overall, 24 (0.9%) clients, and as many as 8 per site (1.7%), were misdiagnosed, with 16 false-positive and 8 false-negative results. Six false-negative specimens were retested with the on-site algorithm on the same sample and were found to be positive. Conversely, 13 false-positive specimens were retested: 8 remained false-positive with the on-site algorithm. CONCLUSIONS: The performance of algorithms at several sites failed to meet expectations and thresholds set by the World Health Organization, with unacceptably high rates of false results. Alongside the careful selection of rapid diagnostic tests and the validation of algorithms, strictly observing correct procedures can reduce the risk of false results. In the meantime, to identify false-positive diagnoses at initial testing, patients should be retested upon initiating antiretroviral therapy.

Towards more accurate HIV testing in sub-Saharan Africa: a multi-site evaluation of HIV RDTs and risk factors for false positives.

INTRODUCTION: Although individual HIV rapid diagnostic tests (RDTs) show good performance in evaluations conducted by WHO, reports from several African countries highlight potentially significant performance issues. Despite widespread use of RDTs for HIV diagnosis in resource-constrained settings, there has been no systematic, head-to-head evaluation of their accuracy with specimens from diverse settings across sub-Saharan Africa. We conducted a standardized, centralized evaluation of eight HIV RDTs and two simple confirmatory assays at a WHO collaborating centre for evaluation of HIV diagnostics using specimens from six sites in five sub-Saharan African countries. METHODS: Specimens were transported to the Institute of Tropical Medicine (ITM), Antwerp, Belgium for testing. The tests were evaluated by comparing their results to a state-of-the-art reference algorithm to estimate sensitivity, specificity and predictive values. RESULTS: 2785 samples collected from August 2011 to January 2015 were tested at ITM. All RDTs showed very high sensitivity, from 98.8% for First Response HIV Card Test 1-2.0 to 100% for Determine HIV 1/2, Genie Fast, SD Bioline HIV 1/2 3.0 and INSTI HIV-1/HIV-2 Antibody Test kit. Specificity ranged from 90.4% for First Response to 99.7% for HIV 1/2 STAT-PAK with wide variation based on the geographical origin of specimens. Multivariate analysis showed several factors were associated with false-positive results, including gender, provider-initiated testing and the geographical origin of specimens. For simple confirmatory assays, the total sensitivity and specificity was 100% and 98.8% for ImmunoComb II HIV 12 CombFirm (ImmunoComb) and 99.7% and 98.4% for Geenius HIV 1/2 with indeterminate rates of 8.9% and 9.4%. CONCLUSION: In this first systematic head-to-head evaluation of the most widely used RDTs, individual RDTs performed more poorly than in the WHO evaluations: only one test met the recommended thresholds for RDTs of ≥99% sensitivity and ≥98% specificity. By performing all tests in a centralized setting, we show that these differences in performance cannot be attributed to study procedure, end-user variation, storage conditions, or other methodological factors. These results highlight the existence of geographical and population differences in individual HIV RDT performance and underscore the challenges of designing locally validated algorithms that meet the latest WHO-recommended thresholds.

Discriminatory capacity between HIV-1 and HIV-2 of the new rapid confirmation assay Geenius.

The currently used HIV confirmatory assays, Western blot and line immunoassay, are costly, complex and time-consuming. There is a need for cheaper, simpler and faster assays for use in high- and low-resource settings. Furthermore, it is necessary to differentiate between HIV-1 and HIV-2 infection due to differences in disease progression, monitoring and treatment options. Because the new Geenius HIV 1/2 Confirmatory Assay (Bio-Rad) has a European Community (CE) label, this study focused on its differentiation capacity using serum/plasma specimens from established HIV-1, HIV-2 and HIV untypable infections from the AIDS Reference Laboratory (ARL) of the Institute of Tropical Medicine (ITM) in Belgium. The results were compared with ARL's standard algorithm for diagnosis of HIV-infection and the new interpretation criteria for discrimination of the INNO-LIA HIVI/II Score, Fujirebio, Ghent, Belgium (LIA). The study showed a performance comparable to that of the reference LIA, with an overall sensitivity of 99.3% and specificity of 98%. Differentiation capacity was much better for the Geenius assay, with 93.8% of samples identified correctly as HIV-1 or HIV-2. When the new interpretation criteria for the LIA were used, the differentiation capacity of LIA increased to 98.5%. The results show that the Geenius assay is a reliable and fast alternative for the confirmation and differentiation of HIV-1 and HIV-2 infection in resource-rich and poor settings.

Use of rapid HIV testing in a low threshold centre in Antwerp, Belgium, 2007-2012.

The Antwerp Helpcenter is a low threshold screening centre for HIV and STI testing focused on high-risk groups. The aim of this work is to describe our experience with the use of rapid HIV tests including the analysis of the characteristics of new cases of HIV infection. We performed a retrospective analysis of all rapid tests routinely performed at the Helpcenter in the period June 2007 to December 2012. The Determine(®)HIV-1/2 (3rd generation) was used until May 2009 and thereafter the Determine Combo(®)HIV-1/2 Ag/Ab (Alere) test (4th generation) on venous blood. All reactive tests were confirmed using a standard confirmation algorithm with ELISAs and a confirmation test (INNO-LIA HIVI/II Score(®)). In all, 5025 rapid tests were performed on blood specimens of 3881 clients including 1173 men having sex with men and 454 migrants from sub-Sahara Africa. The overall prevalence of HIV infection was 1.5% and higher among the risk groups: 4.0% of men having sex with men and 2.2% of migrants from sub-Sahara Africa. The availability of a rapid test was an important reason to present at the Helpcenter. The rapid test was successfully introduced into an outpatient testing centre. Client satisfaction with RT was high and most clients were successfully linked to care.

Performance of a rapid and simple HIV testing algorithm in a multicenter phase III microbicide clinical trial.

A multitest sequential algorithm based on rapid and simple (R/S) assays was applied for the diagnosis of HIV infection among participants in a phase 3 microbicide effectiveness trial. HIV testing was performed on finger-prick blood samples obtained from patients after their enrollment in the trial. The specimens were tested in a serial procedure using three different rapid tests (Determine HIV-1/2 [Abbott], SD Bioline HIV-1/2 3.0 [Standard Diagnostics], and Uni-Gold HIV [Trinity Biotech]). In the event of discordant results between the Determine HIV-1/2 and SD Bioline HIV-1/2 3.0 tests, the third assay (Uni-Gold HIV) determined the final outcome. When the final outcome was positive, a second specimen was collected and tested with the same algorithm, only if a positive result was obtained with this sample the participant was informed of her positive serostatus. A total of 5,734 postenrollment specimens obtained from 1,398 women were tested. Forty-six women tested positive according to the testing algorithm performed on the first collected specimen. Confirmatory testing results obtained at the ITM confirmed that 42 women were truly infected. Two of four initial false positives tested negative upon analysis of a second blood specimen. The other two tested false positive twice using specimens collected the same day. A high percentage of specimens reactive with the Determine HIV-1/2 assay was only observed at the study site in Kampala. This result did not appear to be associated with pregnancy or malaria infection. We conclude that HIV testing algorithms, including only R/S assays, are suitable for use in clinical trials, provided that adequate quality assurance procedures are in place.

Evaluation of four rapid tests for diagnosis and differentiation of HIV-1 and HIV-2 infections in Guinea-Conakry, West Africa.

With both HIV-1 and HV-2 prevalent in Guinea-Conakry, accurate diagnosis and differentiation is crucial for treatment purposes. Thus, four rapid HIV tests were evaluated for their HIV-1 and HIV-2 diagnostic and discriminative capacity for use in Guinea-Conakry. These included SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), Genie II HIV1/HIV2 (Bio-Rad), First Response HIV Card Test 1-2.0 (PMC Medical) and Immunoflow HIV1-HIV2 (Core Diagnostics). Results were compared with gold standard tests (INNO-LIA HIV-I/II Score) and NEW LAV BLOT II (Bio-Rad). Four hundred and forty three sequential stored HIV-positive serum samples, of known HIV-type, were evaluated. Genie II HIV1/HIV2, Immunoflow HIV1-HIV2 and SD Bioline HIV 1/2 3.0 had 100% sensitivity (95% CI, 98.9-100%) while for First Response HIV Card Test 1-2.0 this was 99.5% (95% CI, 98.2%-99.9%). In terms of discriminatory capacity, Genie II HIV1/HIV2 identified 382/ 384(99.5%) HIV-1 samples, 49/ 52(95%) HIV-2 and 7/7(100%) HIV-positive untypable samples. Immunoflow HIV1-HIV2 identified 99% HIV-1, 67% HIV-2 and all HIV-positive untypable samples. First Response HIV Card Test 1-2.0 identified 94% HIV-1, 64% HIV-2 and 57% HIV-positive untypable samples. SD-Bioline HIV 1/2 3.0 was the worst overall performer identifying 65% HIV-1, 69% HIV-2 and all HIV-positive untypable samples. The use of SD Bioline HIV 1/2 3.0 (the current standard in Guinea-Conakry) as a discriminatory HIV test is poor and may be best replaced by Immunoflow HIV1-HIV2.

Obtaining valid laboratory data in clinical trials conducted in resource diverse settings: lessons learned from a microbicide phase III clinical trial.

BACKGROUND: Over the last decade several phase III microbicides trials have been conducted in developing countries. However, laboratories in resource constrained settings do not always have the experience, infrastructure, and the capacity to deliver laboratory data meeting the high standards of clinical trials. This paper describes the design and outcomes of a laboratory quality assurance program which was implemented during a phase III clinical trial evaluating the efficacy of the candidate microbicide Cellulose Sulfate 6% (CS) [1]. METHODOLOGY: In order to assess the effectiveness of CS for HIV and STI prevention, a phase III clinical trial was conducted in 5 sites: 3 in Africa and 2 in India. The trial sponsor identified an International Central Reference Laboratory (ICRL), responsible for the design and management of a quality assurance program, which would guarantee the reliability of laboratory data. The ICRL provided advice on the tests, assessed local laboratories, organized trainings, conducted supervision visits, performed re-tests, and prepared control panels. Local laboratories were provided with control panels for HIV rapid tests and Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) amplification technique. Aliquots from respective control panels were tested by local laboratories and were compared with results obtained at the ICRL. RESULTS: Overall, good results were observed. However, discordances between the ICRL and site laboratories were identified for HIV and CT/NG results. One particular site experienced difficulties with HIV rapid testing shortly after study initiation. At all sites, DNA contamination was identified as a cause of invalid CT/NG results. Both problems were timely detected and solved. Through immediate feedback, guidance and repeated training of laboratory staff, additional inaccuracies were prevented. CONCLUSIONS: Quality control guidelines when applied in field laboratories ensured the reliability and validity of final study data. It is essential that sponsors provide adequate resources for implementation of such comprehensive technical assessment and monitoring systems. TRIAL REGISTRATION: ClinicalTrials.gov NCT00153777 and Current Controlled Trials ISRCTN95638385.