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Background Hepatitis C virus (HCV) screening is critical to HCV elimination efforts. Simplified diagnostics are required for low-resource settings and difficult-to-reach populations. This retrospective study assessed performance of rapid diagnostic tests (RDTs) for detection of HCV antibodies. Methods Two lots of 13 RDTs were evaluated at 3 laboratories using archived plasma samples from 4 countries (Nigeria, Georgia, Cambodia, and Belgium). HCV status was determined using 3 reference tests according to a composite algorithm. Sensitivity and specificity were evaluated in HIV-infected and HIV-uninfected populations. Operational characteristics were also assessed. Results In total, 1710 samples met inclusion criteria. In HIV-uninfected samples (n = 384), the majority of RDTs had sensitivity ≥98% in 1 or both lots and most RDTs had specificity ≥99%. In HIV-infected samples (n = 264), specificity remained high but sensitivity was markedly lower than in HIV-uninfected samples; only 1 RDT reached >95%. The majority of HIV-infected samples for which sensitivity was low did not have detectable HCV viral load/core antigen. Interreader variability, lot-to-lot variability, and rate of invalid runs were low for all RDTs ( Conclusions HCV RDTs should be evaluated in the intended target population, as sensitivity can be impacted by population factors such as HIV status. Clinical Trials Registration NCT04033887

This retrospective study evaluated the reactivity of 3 human immunodeficiency virus (HIV) confirmatory assays (INNO-LIA, Geenius, and MP) and 7 HIV rapid tests on samples from 2 different study populations in Belgium. For the early-treated cohort (83 HIV-1 adult patients treated within 3 months after infection), HIV-1 diagnosis was not obtained in at least 1 confirmatory assay in 12.0% (10/83) and in an HIV rapid test in 31.3% (26/83). Confirmation assay sensitivities ranged from 87.5% to 95.2%, whereas rapid test assay sensitivities ranged from 75.9% to 100%. The time to treatment initiation or the length of time on treatment did not have a statistical influence on the probability to obtain a false-negative test result. The fastest reversion was demonstrated after 4 months of treatment. Among the long-term treated cohort (390 HIV-1 patients with ≥ 9 years of undetectable viral load), false-negative test results were found in at least 1 HIV confirmatory assay for 2.1% (8/390) of the patients and in a HIV rapid test for 4.9% (19/390). Confirmation assay sensitivities ranged from 98.1% to 99.5%, whereas rapid test sensitivities ranged from 96.2% to 100%. Longer treatment increased nonreactivity of the HIV rapid tests (P = .033). Undetectable viral load decreases the sensitivities of HIV diagnostic tests, and further monitoring of the performance of serological assays is advised. ispartof: JOURNAL OF INFECTIOUS DISEASES vol:222 issue:10 pages:1660-1669 ispartof: location:United States status: published

Background: There is today no gold standard method to accurately define the time passed since infection at HIV diagnosis. Infection timing and incidence measurement is however essential to better monitor the dynamics of local epidemics and the effect of prevention initiatives. Methods: Three methods for infection timing were evaluated using 237 serial samples from documented seroconversions and 566 cross sectional samples from newly diagnosed patients: identification of antibodies against the HIV p31 protein in INNO-LIA, SediaTM BED CEIA and SediaTM LAg-Avidity EIA. A multi-assay decision tree for infection timing was developed. Results: Clear differences in recency window between BED CEIA, LAg-Avidity EIA and p31 antibody presence were observed with a switch from recent to long term infection a median of 169.5, 108.0 and 64.5days after collection of the pre-seroconversion sample respectively. BED showed high reliability for identification of long term infections while LAg-Avidity is highly accurate for identification of recent infections. Using BED as initial assay to identify the long term infections and LAg-Avidity as a confirmatory assay for those classified as recent infection by BED, explores the strengths of both while reduces the workload. The short recency window of p31 antibodies allows to discriminate very early from early infections based on this marker. BED recent infection results not confirmed by LAg-Avidity are considered to reflect a period more distant from the infection time. False recency predictions in this group can be minimized by elimination of patients with a CD4 count of less than 100 cells/mm3 or without no p31 antibodies. For 566 cross sectional sample the outcome of the decision tree confirmed the infection timing based on the results of all 3 markers but reduced the overall cost from 13.2 USD to 5.2 USD per sample. Conclusions: A step-wise multi assay decision tree allows accurate timing of the HIV infection at diagnosis at affordable effort and cost and can be an important new tool in studies analyzing the dynamics of local epidemics or the effects of prevention strategies., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Early diagnosis of acute HIV infection can be challenging and is critical in regards to early therapeutic decision making. Objectives To evaluate the performance of different HIV tests in detecting early infections. Study design A 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. Results Four (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. Conclusion RNA 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.

Background Monthly specimens collected from FEM-PrEP—a Phase III trial [1] were investigated for the detection of acute HIV (AHI) infection. Objectives To evaluate the efficiency of the study-specific HIV algorithm in detecting AHI, and the performance of each of the serological and molecular tests used in diagnosing new infections, and their contribution to narrowing the window period. Study design A total of 83 pre-seroconversion specimens from 61 seroconverters from the FEM-PrEP trial were further analyzed in a sub-study. During the trial, HIV seroconversion was diagnosed on site using a testing algorithm with simple/rapid tests (SRTs) and confirmed with a gold standard testing algorithm (see short communication: Fig. 1). The infection date was determined more accurately by the use of standard ELISAs and Nucleic Acid Amplification Tests (NAAT) in a look-back procedure. For this sub-study, the international central laboratory repeated the study algorithm using SRTs. Results A total of 83 pre-seroconversions specimens from 61 seroconverters were analyzed in a look-back procedure. RNA was detected in 35/61 seroconverters at the visit before the seroconversion visit as determined at the study sites. Four seroconversion dates were inaccurate at one study site as the international central laboratory detected the HIV infection one visit earlier using the same test algorithm. Using the gold standard, an additional seroconversion was detected at an earlier visit. The combined antigen/antibody and the single antigen test had a higher sensitivity compared to the SRTs in detecting acute infections. Conclusions In the FEM-PrEP trial, the international central laboratory detected a small number of seroconversions one month earlier than the study sites using the same study algorithm. Standard tests are still the most sensitive tests in detecting pre-seroconversion or acute HIV infection, but they are costly, time consuming and not recommended for use on-site in a clinical trial.

Overtraining, exhaustion, and burnout are widely recognized problems amongst elite athletes. The present research addresses this issue by exploring the extent to which high-quality athlete leadership is associated with elite athletes’ health and burnout. Participants (120 male athletes from three top-division Australian football teams) were asked to rate the quality of each of their teammates in four different leadership roles (i.e., as task and motivational leaders on the field and as social and external leaders off the field), and also to indicate their identification with their team as well as their self-reported health and burnout. Findings indicated that (a) being seen to be a good athlete leader by other members of the team and (b) having a good athlete leader on the team were both positively associated with better team member health and lower burnout. This relationship was mediated by athletes’ identification with their team, suggesting that leaders enhance athletes’ health and reduce athlete burnout by creating and maintaining a sense of shared identity in their team. This, in turn, suggests that coaches can foster an optimal team environment by developing the leadership potential of their athlete leaders – in particular, their skills that foster a sense of shared team identification. This is in the interests not only of team performance but also of team members’ health and burnout. ispartof: EUROPEAN JOURNAL OF SPORT SCIENCE vol:20 issue:7 pages:953-963 ispartof: location:England status: published

This study aimed to analyze the number of task, social and external athlete leaders within sports teams, and to examine the effectiveness of different leadership structures in male and female teams. The participants were 317 male and 214 female soccer players belonging to 18 teams playing in the third highest male division and to 13 teams playing in the highest female division in Spain, respectively. First, we identified the leadership structure in each team (i.e., having zero, one, two or three leaders); second, we grouped the teams according to these leadership structures; and third, MANOVA was used to compare different leadership groups in terms of their effectiveness. The results demonstrated that: (a) the most common structure within the teams was to have one task leader, one social leader, and two external leaders; (b) shared leadership across and within leadership roles was seen as the most effective leadership structure for male and female teams; and (c) male teams showed more benefits when having more task and external leaders, while female teams experienced more benefits when having more task and social leaders on the team. Based on these findings, coaches can optimize their team’s functioning by implementing a structure of shared leadership within their teams, both across and within the different leadership roles. ispartof: PLoS One vol:14 issue:6 pages:e0218167-e0218167 ispartof: location:United States status: published

In this study, we assessed if the superimposition of incident sexually transmitted infections (STIs) on HIV phylogenetic analyses could reveal possible sexual behaviour misclassifications in our HIV-infected population. HIV-1 sequences collected between 1997 and 2014 from 1169 individuals attending a HIV clinic in Antwerp, Belgium were analysed to infer a partial HIV transmission network. Individual demographic, clinical and laboratory data collected during routine HIV follow-up were used to compare clustered and non-clustered individuals using logistic regression analyses. In total, 438 (37.5%) individuals were identified in 136 clusters, including 76 transmission pairs and 60 clusters consisting of three or more individuals. Individuals in a cluster were more likely to have a history of syphilis, Chlamydia and/or gonorrhoea (P

Background Sexually transmitted infections, such as HIV and syphilis, are one of the major health care problems worldwide, especially in low- and middle income countries. HIV screening programmes have been widely used for many years. The introduction of rapid point-of-care tests (RDTs) that can detect both HIV and syphilis, using one single blood specimen, would be a promising tool to integrate the detection of syphilis into HIV programmes and so improve the accessibility of syphilis testing and treatment. Methods As part of the World Health Organization pre-qualification of in vitro diagnostics assessment, the laboratory performance of four dual HIV-Syphilis rapid diagnostic tests (SD Bioline HIV/Syphilis Duo, DPP HIV-Syphilis Assay, Multiplo Rapid TP/HIV Antibody Test and Insti Multiplex HIV-1/HIV-2/Syphilis Antibody Test) was assessed using a well characterized multiregional panel of stored sera specimens. Results In total 400 specimens were tested with each assay, resulting in excellent sensitivities and specificities for HIV, ranging from 99.5 to 100% and from 93.5 to 99.5%, respectively. Results obtained for the Treponema pallidum antibodies were lower, with the lowest sensitivity of 73.5% for Multiplo and the highest of 87% for SD Bioline. Specificities ranged from 99.0 to 100%. Conclusion Although these results suggest that the tests could further improve in accuracy in detection of treponemal antibodies, their introduction into screening programmes to increase the accessibility of HIV/Syphilis diagnosis and treatment for difficult to reach populations in the world is promising.

HIV-1 pol sequences obtained through baseline drug resistance testing of patients newly diagnosed between 2013 and 2017 were analyzed for genetic similarity. For 927 patients the information on genetic similarity was combined with demographic data and with information on the recency of infection. Overall, 48.3% of the patients were genetically linked with 11.4% belonging to a pair and 36.9% involved in a cluster of &ge, 3 members. The percentage of early diagnosed (&le, 4 months after infection) was 28.6%. Patients of Belgian origin were more frequently involved in transmission clusters (49.7% compared to 15.3%) and diagnosed earlier (37.4% compared to 12.2%) than patients of Sub-Saharan African origin. Of the infections reported to be locally acquired, 69.5% were linked (14.1% paired and 55.4% in a cluster). Equal parts of early and late diagnosed individuals (59.9% and 52.4%, respectively) were involved in clusters. The identification of a genetically linked individual for the majority of locally infected patients suggests a high rate of diagnosis in this population. Diagnosis however is often delayed for &gt, 4 months after infection increasing the opportunities for onward transmission. Prevention of local infection should focus on earlier diagnosis and protection of the still uninfected members of sexual networks with human immunodeficiency virus (HIV)-infected members.

Human immunodeficiency virus type 1 (HIV-1) non-B subtype infections occurred in Belgium since the 1980s, mainly amongst migrants and heterosexuals, whereas subtype B predominated in men-having-sex-with-men (MSM). In the last decade, the diagnosis of F1 sub-subtype in particular has increased substantially, which prompted us to perform a detailed reconstruction of its epidemiological history. To this purpose, the Belgian AIDS Reference Laboratories collected HIV-1 pol sequences from all sub-subtype F1-infected patients for whom genotypic drug resistance testing was requested as part of routine clinical follow-up. This data was complemented with HIV-1 pol sequences from countries with a high burden of F1 infections or a potential role in the global origin of sub-subtype F1. The molecular epidemiology of the Belgian subtype F1 epidemic was investigated using Bayesian phylogenetic inference and transmission dynamics were characterized based on birth-death models. F1 sequences were retained from 297 patients diagnosed and linked to care in Belgium between 1988 and 2015. Phylogenetic inference indicated that among the 297 Belgian F1 sequences, 191 belonged to a monophyletic group that mainly contained sequences from people likely infected in Belgium (OR 26.67, 95% CI 9.59–74.15), diagnosed in Flanders (OR 7.28, 95% CI 4.23–12.53), diagnosed at a recent stage of infection (OR 7.19, 95% CI 2.88-17.95) or declared to be MSM (OR 34.8, 95% CI 16.0–75.6). Together with a Spanish clade, this Belgian clade was embedded in the genetic diversity of Brazilian subtype F1 strains and most probably emerged after one or only a few migration events from Brazil to the European continent before 2002. The origin of the Belgian outbreak was dated back to 2002 (95% higher posterior density 2000–2004) and birth-death models suggested that its extensive growth had been controlled (R e < 1) by 2012, coinciding with a time period where delay in antiretroviral treatment initiation substantially declined. In conclusion, phylogenetic reconstruction of the Belgian HIV-1 sub-subtype F1 epidemic illustrates the introduction and substantial dissemination of viral strains in a geographically restricted risk group that was most likely controlled by effective treatment as prevention., SCOPUS: ar.j, info:eu-repo/semantics/published

OBJECTIVES With persisting high numbers of new HIV diagnoses in Europe, HIV testing remains an important aspect of HIV prevention. The traditional centralized and medicalized HIV testing approach has been complemented with newly developed and evaluated non-traditional approaches. Two important factors guided this process: technological innovation and empowerment of the patient. METHODS We present a matrix to develop an HIV testing approach, and elaborate on three commonly used ones: community based testing, self-testing, and self-sampling. Despite non-traditional HIV testing approaches, barriers for testing remain. A potential disadvantage for users is the risk for false-reactive test results. As users receive an orientation test result, a reactive result should be confirmed. Another issue is the window phase, which is longer for some orientation tests compared to a traditional, laboratory-based test. RESULTS Future implementation of non-traditional HIV testing approaches will depend on legal frameworks throughout Europe. Community testing centers may additionally improve empowerment of key populations by expanding their portfolio to testing and treatment for sexually transmitted infections. Community engagement and ownership may imply a shrinking role for health care providers, but they remain crucial actors for personalized information, counselling and referral to specialized HIV-care for many people. CONCLUSIONS A highly effective HIV testing strategy to reduce undiagnosed people living with HIV in Europe is needed. Any approach, chosen according to the principles outlined in this paper, should reach the right people, diagnose them in the most accurate way, and optimize linkage to care.

To improve insight in the drivers of local HIV-1 transmission in Belgium, phylogenetic, demographic, epidemiological and laboratory data from patients newly diagnosed between 2013 and 2015 were combined and analyzed. Characteristics of clustered patients, paired patients and patients on isolated branches in the phylogenetic tree were compared. The results revealed an overall high level of clustering despite the short time frame of sampling, with 47.6% of all patients having at least one close genetic counterpart and 36.6% belonging to a cluster of 3 or more individuals. Compared to patients on isolated branches, patients in clusters more frequently reported being infected in Belgium (95.1% vs. 47.6%; p < 0.001), were more frequently men having sex with men (MSM) (77.9% vs. 42.8%; p < 0.001), of Belgian origin (68.2% vs. 32.9%; p < 0.001), male gender (92.6% vs. 65.8%; p < 0.001), infected with subtype B or F (87.8% vs. 43.4%; p < 0.001) and diagnosed early after infection (55.4% vs. 29.0%; p < 0.001). Strikingly, Sub-Saharan Africans (SSA), overall representing 27.1% of the population were significantly less frequently found in clusters than on individual branches (6.0% vs. 41.8%; p < 0.001). Of the SSA that participated in clustered transmission, 66.7% were MSM and this contrasts sharply with the overall 12.0% of SSA reporting MSM. Transmission clusters with SSA were more frequently non-B clusters than transmission clusters without SSA (44.4% versus 18.2%). MSM-driven clusters with patients of mixed origin may account, at least in part, for the increasing spread of non-B subtypes to the native MSM population, a cross-over that has been particularly successful for subtype F and CRF02_AG. The main conclusions from this study are that clustered transmission in Belgium remains almost exclusively MSM-driven with very limited contribution of SSA. There were no indications for local ongoing clustered transmission of HIV-1 among SSA., SCOPUS: ar.j, info:eu-repo/semantics/published

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. Keywords HIV; diagnostic; test; RDT; misdiagnosis; algorithm (Published: 5 July 2017) Kosack CS et al. Journal of the International AIDS Society 2017, 20 :21419 http://www.jiasociety.org/index.php/jias/article/view/21419 | http://dx.doi.org/10.7448/IAS.20.1.21419

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.

No abstract available. (Published: 19 June 2017) Kosack CS et al. Journal of the International AIDS Society 2017, 20 :22098 http://www.jiasociety.org/index.php/jias/article/view/22098 | http://dx.doi.org/10.7448/IAS.20.1.22098

publishersversion published

There is need for more evaluations of non-invasive tests in order to broaden the reach of testing programs and to perform large scale epidemiological studies. In this study, three different human immunodeficiency virus (HIV) enzyme linked immunosorbent assays (ELISAs) and one line immunoassay were evaluated to detect HIV antibodies in oral fluid samples. Specimens were collected, after informed consent was obtained, with the Oracol (MMD, Worcester, England) device. A total IgG quantitation test was performed to demonstrate the quality of the sample. Assessment of a modified protocol of the Vironostika® HIV Ag/Ab, Enzygnost® Anti-HIV 1/2 Plus Genscreen™ HIV-1/2 Version 2 and a line immune confirmatory assay the INNO-LIA™ HIV I/II score was done, using oral fluid specimens of 325 HIV positive and negative individuals. For the ELISAs, the addition of an extra internal oral fluid control was evaluated as well as different cut-offs, time between sampling and testing and the effect of drinking water just before sampling. Finally, the confirmatory test and some testing algorithms and combination of tests were discussed. The results obtained from the oral fluid specimens were compared with the gold standard on paired serum specimens. Firstly, there was no significant difference observed between the use of the kit controls and the oral fluid controls. New protocols and calculation of cut-offs were defined for two of the three ELISAs. High sensitivities and specificities were obtained with all three ELISAs without any statistical difference between the three tests. Secondly, no statistically significant difference was observed when samples were stored for different time periods between sampling and testing, meaning that a period of seven days at room temperature before testing is still acceptable. Thirdly, drinking water before sample collection did not interfere with the testing, although lower optical densities were observed. None of the positive samples were missed. In addition, the line immunoassay INNO-LIA™ HIV I/II score test is a promising test for confirmation of reactive oral fluid specimen, but more samples need to be validated in order to adapt the interpretation rules specifically for oral fluid specimens. Different choices/algorithms adapted for the purpose of testing can be proposed. In conclusion, it can be said that the commercial ELISAs with adapted protocol and cut-off values are suitable tools for making HIV test performance accessible to people. With this non-invasive sampling method, more eligible individuals can and will be selected for further HIV test on blood.

BACKGROUND: The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPC) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence. METHODS:: Ten HIV+ adults with HIV RNA

Next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of rapid between- and within-host evolution may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions.De novoassembly avoids this bias by effectively aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the toolshiverto preprocess reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We useshiverto reconstruct the consensus sequence and minority variant information from paired-end short-read data produced with the Illumina platform, for 65 existing publicly available samples and 50 new samples. We show the systematic superiority of mapping toshiver’s constructed reference over mapping the same reads to the standard reference HXB2: an average of 29 bases per sample are called differently, of which 98.5% are supported by higher coverage. We also provide a practical guide to working with imperfect contigs.

Background: Late human immunodeficiency virus (HIV) diagnosis is common among sub-Saharan African migrants. To address their barriers to HIV testing uptake and improve timely HIV diagnoses and linkage to care, the outreach HIV testing intervention, “swab2know,” was developed. It combined a community-based approach with innovative testing methods: oral fluid self-sampling and the choice between Web-based HIV test result collections using a secured website or post-test counseling at a sexual health clinic. The sessions included an informational speech delivered by a physician of sub-Saharan African origin and testimonies by community members living with HIV. Objectives: The objectives of this study were to evaluate the intervention’s acceptability among sub-Saharan African migrants and its potential to reach subgroups at higher risk for HIV infection and to identify facilitators and barriers for HIV testing uptake. Methods: This mixed-method study combined qualitative (participant observations and informal interviews with testers and nontesters) and quantitative data (paper–pencil survey, laboratory data, and result collection files). Data were analyzed using a content analytical approach for qualitative and univariate analysis for quantitative data. Results: A total of 10 testing sessions were organized in sub-Saharan African migrant community venues in the city of Antwerp, Belgium, between December 2012 and June 2013. Overall, 18.2% of all people present (N=780) underwent HIV testing; 29.8% of them tested for HIV for the first time, 22.3% did not have a general practitioner, and 21.5% reported 2 or more sexual partners (last 3 months). Overall, 56.3% of participants chose to collect their HIV test results via the protected website. In total, 78.9% collected their results. The qualitative analysis of 137 participant observation field notes showed that personal needs and Internet literacy determined the choice of result collection method. Generally, the oral fluid collection devices were well accepted mainly because sub-Saharan African migrants dislike blood taking. For some participants, the method raised concerns about HIV transmission via saliva. The combination of information sessions, testimonies, and oral fluid collection devices was perceived as effectively reducing thresholds to participation. Acceptability of the intervention differed between individual participants and settings. Acceptance was higher among women, in churches and settings where community leaders were engaged in HIV awareness raising. Higher preventive outcomes were observed in settings with lower acceptance. The presence of the intervention team visualized the magnitude of the HIV epidemic to the public and promoted HIV testing uptake at large, for example, those who declined indicated they would take up testing later. Conclusions: When accompanied by tailored provision of information, outreach HIV testing interventions adopting a community-based approach and innovative methods such as Web-based result collection and oral fluid collection devices are acceptable and reduce thresholds for HIV testing uptake. The swab2know intervention was able to reach sub-Saharan African migrants at risk of HIV infection, and with limited access to regular HIV testing. Among nontesters, the intervention contributed to awareness raising and therefore has a place in a multipronged HIV test promotion strategy. [JMIR Public Health Surveill 2016;2(2):e33]

BACKGROUND: In the wake of the recent outbreak of Ebola virus disease (EVD) in several African countries, the World Health Organization prioritized the evaluation of treatment with convalescent plasma derived from patients who have recovered from the disease. We evaluated the safety and efficacy of convalescent plasma for the treatment of EVD in Guinea. METHODS: In this nonrandomized, comparative study, 99 patients of various ages (including pregnant women) with confirmed EVD received two consecutive transfusions of 200 to 250 ml of ABO-compatible convalescent plasma, with each unit of plasma obtained from a separate convalescent donor. The transfusions were initiated on the day of diagnosis or up to 2 days later. The level of neutralizing antibodies against Ebola virus in the plasma was unknown at the time of administration. The control group was 418 patients who had been treated at the same center during the previous 5 months. The primary outcome was the risk of death during the period from 3 to 16 days after diagnosis with adjustments for age and the baseline cycle-threshold value on polymerase-chain-reaction assay; patients who had died before day 3 were excluded. The clinically important difference was defined as an absolute reduction in mortality of 20 percentage points in the convalescent-plasma group as compared with the control group. RESULTS: A total of 84 patients who were treated with plasma were included in the primary analysis. At baseline, the convalescent-plasma group had slightly higher cycle-threshold values and a shorter duration of symptoms than did the control group, along with a higher frequency of eye redness and difficulty in swallowing. From day 3 to day 16 after diagnosis, the risk of death was 31% in the convalescent-plasma group and 38% in the control group (risk difference, -7 percentage points; 95% confidence interval [CI], -18 to 4). The difference was reduced after adjustment for age and cycle-threshold value (adjusted risk difference, -3 percentage points; 95% CI, -13 to 8). No serious adverse reactions associated with the use of convalescent plasma were observed. CONCLUSIONS: The transfusion of up to 500 ml of convalescent plasma with unknown levels of neutralizing antibodies in 84 patients with confirmed EVD was not associated with a significant improvement in survival. (Funded by the European Union's Horizon 2020 Research and Innovation Program and others; ClinicalTrials.gov number, NCT02342171.). ispartof: New England Journal of Medicine vol:374 issue:374 pages:33-42 ispartof: location:United States status: published

Background: Sub-Saharan African Migrants (SAM) are the second largest group affected by HIV/AIDS in Belgium and the rest of Western Europe. Increasing evidence shows that, more than previously thought, SAM are acquiring HIV in their host countries. This calls for a renewed focus on primary prevention. Yet, knowledge on the magnitude of the HIV epidemic among SAM (HIV prevalence estimates and proportions of undiagnosed HIV infections) and underlying drivers are scarce and limit the development of such interventions. Objective: By applying a community-based participatory and mixed-methods approach, the TOGETHER project aims to deepen our understanding of HIV transmission dynamics, as well as inform future primary prevention interventions for this target group. Methods: The TOGETHER project consists of a cross-sectional study to assess HIV prevalence and risk factors among SAM visiting community settings in Antwerp city, Belgium, and links an anonymous electronic self-reported questionnaire to oral fluid samples. Three formative studies informed this method: (1) a social mapping of community settings using an adaptation of the PLACE method; (2) a multiple case study aiming to identify factors that increase risk and vulnerability for HIV infection by triangulating data from life history interviews, lifelines, and patient files; and (3) an acceptability and feasibility study of oral fluid sampling in community settings using participant observations. Results: Results have been obtained from 4 interlinked studies and will be described in future research. Conclusions: Combining empirically tested and innovative epidemiological and social science methods, this project provides the first HIV prevalence estimates for a representative sample of SAM residing in a West European city. By triangulating qualitative and quantitative insights, the project will generate an in-depth understanding of the factors that increase risk and vulnerability for HIV infection among SAM. Based on this knowledge, the project will identify priority subgroups within SAM communities and places for HIV prevention. Adopting a community-based participatory approach throughout the full research process should increase community ownership, investment, and mobilization for HIV prevention.

HIV subtype-specific data on mutation type, rate, and accumulation following HAART treatment failure are limited. We studied patterns and accrual of drug resistance mutations in a Cambodian CRF01_AE-infected cohort continuing a virologically failing first-line, nonnucleoside reverse transcriptase inhibitor- (NNRTI-) based, HAART. Between 2005 and 2007, 837 adult HIV-infected patients had regular plasma HIV-1 RNA viral load measurements at Sihanouk Hospital Centre of Hope (SHCH), Cambodia. Drug resistance testing was performed in all patients with HIV-1 RNA1000 copies/ml after at least 6 months of HAART. Seventy-one patients with a mean age of 34 years, of whom 68% were male, were retrospectively assessed at virological failure. The median duration of antiretroviral therapy was 12.3 (IQR 7.1-18.23) months, the median CD4 cell count was 173 (IQR 118-256) cells/mm(3), and the mean plasma HIV-1 RNA viral load was 3.9 log (SD 0.72) at failure. NNRTI mutations, M184I/V mutation, thymidine analogue mutations, and K65R were observed in 78.9%, 69%, 20%, and 12.7% of patients, respectively. For 33 patients, genotypic testing was carried out on at least two occasions before the switch to second-line HAART after a median duration of 5.8 (IQR 4.3-6.1) months of virological failure: 54.5% of patients accumulated new mutations with a rate of 1.6 mutations per person-year. Accumulation was seen both for nucleoside and nonnucleoside reverse transcriptase inhibitors, and also in patients with low-level viremia. Subtype-specific data on mutation type, rate, and accumulation after HAART failure are urgently needed to optimize treatment strategies in resource-limited settings.

The accuracy of diagnostic tests for HIV in patients with tropical infections is poorly documented. Human African trypanosomiasis (HAT) is characterized by a polyclonal B-cell activation, constituting a risk for false-positive reactions to diagnostic tests, including HIV tests. A retrospective study of the accuracy of HIV diagnostic tests was performed with 360 human African HAT patients infected with Trypanosoma brucei gambiense before treatment and 163 T. b. gambiense -infected patients 2 years after successful treatment in Mbuji Mayi, East Kasai, Democratic Republic of the Congo. The sensitivities, specificities, and positive predictive values (PPVs) of individual tests and algorithms consisting of 3 rapid tests were determined. The sensitivity of all tests was 100% (11/11). The low specificity (96.3%, 335/348) and PPV (45.8%, 11/24) of a classical seroconfirmation strategy (Vironostika enzyme-linked immunosorbent assay [ELISA] followed by line immunoassay) complicated the determination of HIV status, which had to be determined by PCR. The specificities of the rapid diagnostic tests were 39.1% for Determine (136/348); 85.3 to 92.8% (297/348 to 323/348) for Vikia, ImmunoFlow, DoubleCheck, and Bioline; and 96.6 to 98.3% (336/348 to 342/348) for Uni-Gold, OraQuick, and Stat-Pak. The specificity of Vironostika was 67.5% (235/348). PPVs ranged between 4.9 and 64.7%. Combining 3 different rapid tests resulted in specificities of 98.3 to 100% (342/348 to 348/348) and PPVs of 64.7 to 100% (11/17 to 11/11). For cured HAT patients, specificities were significantly higher for Vironostika, Determine, Uni-Gold, and ImmunoFlow. T. b. gambiense infection decreases the specificities of antibody detection tests for HIV diagnosis. Unless tests have been validated for interference with HAT, HIV diagnosis using classical algorithms in untreated HAT patients should be avoided. Specific, validated combinations of 3 HIV rapid tests can increase specificity.

BACKGROUND: The recent introduction of highly sensitive viral load assays resulted in a significant increase in number of treated HIV-infected patients with a detectable viral load. The significance of a viral load between 20 and 50 copies/mL remains unclear. OBJECTIVES: To compare the performance of three viral load assays, with special attention for specificity and sensitivity at the lowest level of quantification. STUDY DESIGN: Samples (n=181) were selected from 62 HIV-positive individuals that experience viral blips or episodes of low but detectable viremia under antiretroviral treatment, and from 216 HIV-negative individuals. Each sample was tested in at least two of three assays: the Cobas Amplicor HIV-1 Monitor (CAP/CA), the Cobas Ampliprep/Cobas TaqMan HIV-1 version 1 (CAP/CTM1) and the Cobas Ampliprep/Cobas TaqMan HIV-1 version 2 (CAP/CTM2). RESULTS: No false positive results were recorded. Kappa statistics revealed fair to moderate agreement between the results of the three assays, but important differences in sensitivity were observed, with the highest sensitivity reported for CAP/CTM2 followed by CAP/CTM1 and CAP/CA. The differences in sensitivity remained after equalization of the detection limit for all assays at 50 copies/mL. Analysis of samples collected over time showed that patients with single blips in CAP/CA present with recurrent blips in CAP/CTM1 and persistent detectable viremia in CAP/CTM2. CONCLUSIONS: Viral load results between 20 and 50 copies/mL in either CAP/CTM1 or CAP/CTM2, indicate true viremia. The availability of highly sensitive assays force reconsideration of the terms 'undetectable' viral load and 'virological success' of antiretroviral treatment. Copyright 2010 Elsevier B.V. All rights reserved.

The intrasplenic injection of human peripheral blood mononuclear cells (PBMCs) into severely immune deficient NOD/SCID mice, causes a massive and transient dominant expansion of human B cells in the spleen. This permits the easy isolation of human monoclonal antibodies specific for different antigens by a Kohler and Milstein-based method. Here we studied the human HIV-specific antibody response in the circulation of mice after intrasplenic transfer of PBMC from untreated HIV-infected patients with detectable to high viral load as well as from HAART-treated and from untreated patients, who kept an undetectable viral load (the latter referred to as “natural suppressors”). Excellent B cell expansion was obtained for all PBMC. High level replication of virus was observed after transfer of PBMC of untreated viremic patients only. A strong and multispecific HIV-specific antibody response was observed after transfer of PBMC of untreated viremic patients and natural suppressors. In contrast, only a weak and pauci-specific antibody response was detected in mice reconstituted with PBMC from successfully treated patients. Based on these observations we conclude that the use of the intraspleen mouse model confirmed a) the presence of HIV-specific circulating memory B cells in untreated patients and natural suppressors; b) the nearly complete absence of circulating memory B cells in patients receiving highly active antiretroviral therapy. Using the intraspleen model we generated large numbers of immortalized B cells and isolated two anti-p24 human monoclonal antibodies. We further conclude that the intraspleen huPBL NOD/SCID model is a small animal model useful for the analysis of the antibody response against HIV found in patients.

Background: Non-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete. Methods: Pol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering. Results: The majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM. Conclusions: The extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero-as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored.

Oral fluid has many advantages over blood-based techniques: it is less invasive, eliminates the occupational risk associated with needle stick accidents and collection can be self-administrated. Each individual test is packaged with a corresponding collection device. This study tested the suitability of the Intercept Oral Specimen Collection Device for different HIV diagnostic tests: three different rapid HIV tests and two adapted ELISAs, which were evaluated and compared with a gold standard on blood. In addition a total IgG quantification was performed to demonstrate the quality of the specimen. HIV antibodies were detected with a sensitivity of 100%, 99.3%, 98.6%, 100% and 95.7% for, DPP, OraQuick, Aware, Genscreen and Vironostika respectively using the Intercept Collection Device. Respective specificities were 100%, 100%, 99.3%, 97.3% and 100%.

Background: As HIV remains a public health concern, increased testing among those at risk for HIV acquisition is important. Men who have sex with men (MSM) are the most important group for targeted HIV testing in Europe. Several new strategies have been developed and implemented to increase HIV-testing uptake in this group, among them the Swab2know project. Objective: In this project, we aim to assess the acceptability and feasibility of outreach and online HIV testing using oral fluid samples as well as Web-based delivery of test results. Methods: Sample collection happened between December 2012 and April 2014 via outreach and online sampling among MSM. Test results were communicated through a secured website. HIV tests were executed in the laboratory. Each reactive sample needed to be confirmed using state-of-the-art confirmation procedures on a blood sample. Close follow-up of participants who did not pick up their results, and those with reactive results, was included in the protocol. Participants were asked to provide feedback on the methodology using a short survey. Results: During 17 months, 1071 tests were conducted on samples collected from 898 men. Over half of the samples (553/1071, 51.63%) were collected during 23 outreach sessions. During an 8-month period, 430 samples out of 1071 (40.15%) were collected from online sampling. Additionally, 88 samples out of 1071 (8.22%) were collected by two partner organizations during face-to-face consultations with MSM and male sex workers. Results of 983 out of 1071 tests (91.78%) had been collected from the website. The pickup rate was higher among participants who ordered their kit online (421/430, 97.9%) compared to those participating during outreach activities (559/641, 87.2%; P

In order to evaluate alternative tests and strategies to simplify pediatric human immunodeficiency virus (HIV) screening at the district hospital level, a cross-sectional exploratory study was organized in the Democratic Republic of the Congo. Venous and capillary phlebotomies were performed on 941 Congolese children, aged 1 month to 12 years (153 children under 18 months and 788 children more than 18 months old). The HIV prevalence rate was 4.7%. An algorithm for children more than 18 months old, using serial rapid tests (Determine, InstantScreen, and Uni-Gold) performed on capillary blood stored in EDTA tubes, had a sensitivity of 100.0% (95% confidence interval [CI], 88.9 to 100.0%) and a specificity of 100.0% (95% CI, 99.5 to 100.0%). The results of this study suggest that the ultrasensitive p24 antigen assay may be performed on capillary plasma stored on filter paper (sensitivity and specificity, 100.0%;n= 87) instead of venous plasma (sensitivity, 92.3%; specificity, 100.0%;n= 150). The use of glucolets (instruments used to perform capillary phlebotomies), instead of syringes and needles, may reduce procedural pain and the risk of needle stick injuries at a comparable cost. Compared to the reference, HIV could have been correctly excluded based on one rapid test for at least 90% of these children. The results of this study point towards underutilized opportunities to simplify phlebotomy and pediatric HIV screening.

The protective role of beta-chemokines in HIV infection and disease remains controversial. Contradictory findings have been reported possibly as the result of different beta-chemokine detection methods. To test this, peripheral blood lymphocytes from treatment-naive HIV patients, patients on highly active antiretroviral therapy (HAART), and uninfected controls were assessed for intracellular beta-chemokine levels in comparison with levels of beta-chemokine secretion in culture supernatants. HIV patients had significantly higher intracellular levels of macrophages inflammatory protein (MIP)-1 alpha and MIP-1 beta than uninfected control subjects. In contrast, MIP-1 alpha and MIP-1 beta supernatant levels were significantly lower in HIV patients than in controls. Interestingly, both intracellular and supernatant levels of RANTES (regulated on activation, normal T cell expressed and secreted) were significantly increased in HIV patients. Prolonged (> 3 years) administration of HAART in HIV patients normalized the intracellular levels of MIP-1 beta and RANTES and restored the decreased supernatant levels of MIP-1 alpha and MIP-1 beta to levels observed among controls. Significant direct correlations observed between the intracellular and the supernatant levels of beta-chemokines in controls were lost in treatment-naive (except MIP-1 beta) and HAART-treated patients (except RANTES after 3 years of HAART). These data indicate that lymphocytes of HIV patients display a disrupted capacity to secrete the beta-chemokines MIP-1 alpha and MIP-1 beta, which may constitute a mechanism of immune dysfunction in progressive HIV infection. Furthermore, we demonstrated that the detection of beta-chemokines in HIV patients by different methods may indeed result in contradictory findings.

Monitoring the efficacy of highly active antiretroviral treatment (HAART) is crucial if disease progression and the emergence of viral mutants are to be avoided. Classical viral load monitoring is too expensive for large-scale use in resource-limited settings. Three alternative measures, CD4 count, total lymphocyte count (TLC) and haemoglobin, were evaluated as surrogate markers of treatment success (viral load below detection level) among 710 HIV-positive patients who started HAART in an HIV treatment centre in Belgium. TLC correlated well with changes in CD4 counts during HAART, but an increase in TLC alone was a poor predictor of treatment success. A combination of increases in both haemoglobin levels and TLC proved a reliable predictor of successful treatment outcome comparable to the increase in CD4 count, but its specificity and sensitivity were low.

Viral fitness can be modified upon development of antiretroviral drug resistance, usually by selection of compensatory mutations. In this study, we have used HIV-1 isolates from individuals receiving a protease inhibitor (PI)-based regimen to analyze the impact of basal genetic background on viral fitness evolution. Paired plasma samples and HIV-1 isolates were obtained from 10 PI-naive HIV-infected individuals enrolled in 2 different studies of combination antiretroviral therapy. Genomic regions from pal and env were sequenced. Viral fitness was measured using growth competition experiments followed by heteroduplex tracking analysis. Baseline genotypic analyses of pol showed that 9 of 10 viruses had a different degree of secondary mutations in the protease gene at codons associated with PI resistance (i.e., 101, 361, 63P, 71T, and 771). After 48 weeks of PI-based therapy, a strong correlation was observed between protease genetic divergence and viral fitness difference (r = 0.78, P = 0.03), but not with reverse transcription or Env divergence, suggesting that genotypic changes in the protease gene were driving HIV-1 evolution in these patients. As expected, an inverse correlation was observed between the number of protease and reverse transcription primary mutations and viral fitness (r = -0.65, P < 0.0001). However, our results suggest that the preexistence of secondary mutations in protease genetic background may have implications in HIV-1 fitness evolution and virologic response to antiretroviral therapy.

Because prevention of heterosexual HIV transmission is not always possible, it is important to develop effective strategies of postexposure prophylaxis (PEP). Since in vivo comparison of drug potency is difficult, we developed an in vitro model with cells resembling primary targets during sexual transmission: monocyte-derived dendritic cells (MO-DCs), Langerhans cells (MO-LCs), and resting autologous CD4(+) T cells. Nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively) were evaluated for their antiviral activity, when added immediately after infection or at a later time point. In parallel, their immune-suppressive effect was examined by measuring inhibition of mixed MO-DC/allogeneic CD4(+) T cell cultures. Most RTIs potently inhibited HIV replication, even if added 24 hr after infection (representing PEP). The sensitivity to antiretroviral drugs was similar in HIV-infected MO-DCs and MO-LCs, but decreased in cocultures with resting autologous CD4(+) T cells. The NNRTIs efavirenz and UC-781 as well as the NRTIs AZT, 3TC, and d4T showed a similar high potency in MO-DC plus autologous CD4(+) T cell cocultures as compared with CEM T cells, whereas their activity in phytohemagglutinin/interleukin 2 (PHA/IL-2)-activated CD4(+) T cells was lower. The dideoxynucleoside RTI abacavir as well as the phosphonates (R)-PMPA and PMEA were more active in infected MO-DCs as compared with either CEM T cells or PHA/IL-2 activated CD4(+) T cells. Infection in cocultures of MO-DCs and autologous CD4(+) T cells could be aborted in a proportion of the cultures, with high concentrations of PMEA and/or efavirenz, but not with AZT. Suppressive activity in mixed leukocyte cultures was observed only at very high concentrations of RTI. Our data suggest that cocultures of MO-DCs and autologous CD4(+) T cells can be used as a possible in vitro model to explore protocols for PEP after sexual HIV transmission.

To evaluate the performance of 22 assays for the detection of antibodies to HIV. Twenty-two assays for the combined detection of antibodies to HIV-1 and HIV-2, were evaluated on the same panel of serum specimens of diverse origin. Eight of the assays were ELISAs and the remaining 14 were simple, assays read visually. The specimen panel consisted of anti-HIV positive and negative samples from Africa (n=192), Europe (n=206), Asia (n=99) and Latin America (n=98). In addition to estimations of sensitivity and specificity, the assays were assessed, using a novel scoring system, for their ease of performance and for their suitability for use in small laboratories and clinics. The sensitivities of the assays in terms of seroconversion were assessed using series of specimens collected from nine individuals undergoing seroconversion. Eight ELISAs and eight of 14 simple assays had sensitivities and specificities of >99 and 95%, respectively. The results of these evaluations will be of assistance to those responsible for the selection of appropriate anti-HIV assays according to laboratory circumstances, the purpose of the testing and the population being tested.

Background: Pre-exposure prophylaxis (PrEP) is a promising and effective tool to prevent HIV. With the approval of Truvada as daily PrEP by the European Commission in August 2016, individual European Member states prepare themselves for PrEP implementation following the examples of France and Norway. However, context-specific data to guide optimal implementation is currently lacking. Objective: With this demonstration project we evaluate whether daily and event-driven PrEP, provided within a comprehensive prevention package, is a feasible and acceptable additional prevention tool for men who have sex with men (MSM) at high risk of acquiring HIV in Belgium. The study’s primary objective is to document the uptake, acceptability, and adherence to both daily and event-driven PrEP, while several secondary objectives have been formulated including impact of PrEP use on sexual behavior. Methods: The Be-PrEP-ared study is a phase 3, single-site, open-label prospective cohort study with a large social science component embedded in the trial. A total of 200 participants choose between daily or event-driven PrEP use and may switch, discontinue, or restart their regimen at the 3-monthly visits for a duration of 18 months. Data are collected on several platforms: an electronic case report form, a Web-based tool where participants register their sexual behavior and pill use, a more detailed electronic self-administered questionnaire completed during study visits on a tablet computer, and in-depth interviews among a selected sample of participants. To answer the primary objective, the recruitment rate, (un)safe sex behavior during the last 6 months, percentage of reported intention to use PrEP in the future, retention rates in different regimens, and attitudes towards PrEP use will be analyzed. Adherence will be monitored using self-reported adherence, pill count, tenofovir drug levels in blood samples, and the perceived skills to adhere. Results: All participants are currently enrolled, and the last study visit is planned to take place around Q3 2018. Conclusions: As PrEP is not yet available in Belgium for use, this study will provide insights into how to optimally implement PrEP within the current health care provision and will shape national and European guidelines with regard to the place of PrEP in HIV prevention strategies. ClinicalTrial: EU Clinical Trial 2015-000054-37; https://www.clinicaltrialsregister.eu/ctr-search/trial/2015-000054-37/BE (Archived by WebCite at http://www.webcitation.org/6nacjSdmM).

Background Pre-exposure prophylaxis (PrEP) with daily oral emtricitabine (FTC)/tenofovir disoproxil fumarate may select for drug resistance if there is low adherence. Methods Plasma viral HIV-1 RNA level, CD4+ T-cell counts, and drug resistance were evaluated among seroconverting women in the FEM-PrEP trial (clinicaltrials.gov NCT00625404) using standard clinical tests, allele-specific PCR (ASPCR), and by deep sequencing. Tenofovir, FTC, and their intracellular metabolites were measured in plasma and cells. Results There was no difference in plasma HIV-1 RNA level or CD4+ cell count among seroconverters in the active arm versus those receiving placebo. Tenofovir resistance was not observed. FTC resistance was detected using clinical assays in five seroconverters (four in the active arm and one in the placebo arm); two in the active arm occurred among women having moderate concentrations of PrEP drugs in the blood. The first evidence of infection occurred at the first postenrollment visit in three of the four with FTC resistance, although none had detectable viral nucleic acids at enrollment. FTC-resistant minor variants were detected in an additional four seroconverters (one in the active arm and three in the placebo arm). Conclusions Drug resistance detected during ineffective PrEP use had characteristics suggesting transmitted infection or incubating infection prior to starting PrEP.

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1). To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. Conclusions: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

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.

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.

BACKGROUND Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Introduction : We studied factors associated with the continuum of HIV care in Belgium. Methods : Data of the national registration of new HIV diagnosis and of the national cohort of HIV-infected patients in care were combined to obtain estimates of and factors related with proportions of HIV-infected patients in each step of the continuum of care from diagnosis to suppressed viral load (VL). Factors associated with ignorance of HIV seropositivity were analyzed among patients co-infected with HIV and STI in the Belgian STI sentinel surveillance network. Associated factors were identified by multivariate logistic regression. Results : Among 4038 individuals diagnosed with HIV between 2007 and 2010, 90.3% were linked to care. Of 11684 patients in care in 2010, 90.8% were retained in care up to the following year, 88.3% of those were on ART, of whom 95.3% had suppressed VL (

In a longitudinal study we address the hypothesis that resis tance to disease progression in lentivirus-infected chimpanzees is related to potent non-cytotoxic suppression of virus replication. In a long-term follow-up, the viral suppressive capacity in two simian immunodeficiency virus (SIV)cpz-infected chimpanzees was correlated with two polymerase chain reaction (PCR)- and two culture-based virus load measurements. In both animals, quantitative virus isolation (QVI) tended to decline slowly, whereas in vitro virus suppression was sustained or increased over time. In general, plasma virus loads in SIVcpz-infected animals were maintained for extended periods of time. Based on current assays that measure virus suppressive capacity in peripheral blood, it was not possible to conclude that virus suppression played a major role in the maintenance of the disease-free state in lentivirus-infected chimpanzees.