Affordable blood culture systems from China: in vitro evaluation for use in resource-limited settings.

BACKGROUND: Bloodstream infections (BSI) pose a significant threat due to high mortality rates and the challenges posed by antimicrobial resistance (AMR). In 2019, an estimated 4.95 million deaths were linked to bacterial AMR. The highest impact was seen in resource-limited settings (RLS). For diagnosis of BSI, performant continuously-monitoring blood culture systems (CMBCS) have been optimized. However, in RLS, the implementation of CMBCS is hindered by budget constraints and unsuitable environmental conditions. Manufacturers from growing economies are currently producing affordable in vitro diagnostics, which could fill the gap in capacity, but so far these are not established outside their domestic markets. METHODS: This study evaluated the performance, usability, and interchangeability of Chinese CMBCS in a laboratory setting using simulated blood cultures with a panel of 20 BSI-associated strains. Four systems were selected for the assessment: Autobio BC60, Mindray TDR60, Scenker Labstar50, and DL-biotech DL-60. FINDINGS: Overall, all evaluated CMBCS demonstrated good performance with high yield (96.7-100%) and specificity (97.5-100%), comparable to the reference system (bioMérieux 3D). In addition, when used as "manual" blood cultures in a conventional incubator with visual growth detection, performance was also satisfactory: yield was between 90 and 100% and specificity was 100% for all BCBs. Both the CMBCS and the BCBs were easy to use and lot-to-lot variability in BCBs was minimal. The interchangeability testing indicated that the BCBs from different brands (all except Scenker) were compatible with the various automates, further highlighting the potential for a harmonized "universal BCB." INTERPRETATION: Based on this in vitro study, we recommend the use of these systems in settings with challenging environments and limited resources. The Autobio system performed best for automatic detection and DL-Biotech BCBs for manual cultures respectively (combination of performance, price, usability). The appropriateness for use in RLS should still be confirmed in a field study. FUNDING: The study was funded by FIND.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Acceptability of a Community-Based Outreach HIV-Testing Intervention Using Oral Fluid Collection Devices and Web-Based HIV Test Result Collection Among Sub-Saharan African Migrants: A Mixed-Method Study.

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.

Swab2know: An HIV-Testing Strategy Using Oral Fluid Samples and Online Communication of Test Results for Men Who Have Sex With Men in Belgium.

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<.001). MSM participating during outreach activities versus online participants were more likely to have never been tested before (17.3% vs 10.0%; P=.001) and reported more sexual partners in the 6 months prior to participation in the project (mean 7.18 vs 3.23; P<.001). A total of 20 participants out of 898 (2.2%) were confirmed HIV positive and were linked to care. Out of 1071 tests, 28 (2.61%) with a weak reactive result could not be confirmed, and were thereby classified as false reactive results. Most of the 388 participants who completed posttest surveys (388/983, 39.5%) were very positive about their experience. The vast majority (371/388, 95.6%) were very satisfied, while 17 out of 388 (4.4%) reported mixed feelings. CONCLUSIONS: Despite a high yield and a considerable number of false reactive results, satisfaction was high among participants. The project helped us to reach the target population, both in numbers of tests executed and in newly diagnosed HIV infections. Further optimization should be considered in the accuracy of the test, the functionalities of the website (including an online counseling tool), and in studying the cost effectiveness of the methodology.

Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays.

HIV-1 Env pseudotyped viruses (PV) are an attractive tool for studying the antiviral activities of compounds interfering with virus entry into a target cell. To investigate whether results obtained in PV assays are relevant biologically, the antiviral activity of 6 reference compounds was compared on 5 virus isolates of different clades using three assays: (1) replicating virus in peripheral blood mononuclear cells (PBMCs), (2) PV in CD4 and CCR5- or CXCR4 co-receptor expressing Ghost cells, and (3) PV in PBMCs. A significant linear relationship was found between both single-cycle PV assays (P<0.0001, R2=0.75). Moreover, both assays showed enhanced sensitivity to the antiretrovirals tested (P=0.013 and 0.015, respectively) as compared to the PBMC assay with replication-competent virus. Most importantly, results from the latter assay could be predicted significantly from both PV assays, in which either Ghost target cells (P<0.0001, R2=0.61) or PBMCs (P<0.0001, R2=0.55) were used. The usefulness of the PV assay was demonstrated further by investigating the impact of the HIV-1 Env subtype on the antiviral activity of five new compounds derived from the entry inhibitor BMS806.

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.

Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients.

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates.

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.

The first generation of candidate HIV-1 vaccines can induce antibodies able to neutralize primary isolates in assays with extended incubation phases.

Quantification of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies is considered to be an important parameter in evaluating candidate vaccines. Most previous studies have failed to detect vaccine-induced antibodies against primary isolates, which are more resistant to antibody mediated neutralization compared with laboratory isolates, in neutralization assays. In this study, sera from a prime boost vaccination strategy of a phase I clinical trial were tested against six clade B primary HIV-1 isolates and single isolates of clades C and F. These sera produced statistically significant neutralization against primary isolates MN, SF13, SF162 and Han 2 but not the most resistant subtype B isolates (92US077 and 93US143) nor the subtype C and F isolates. These data suggest that the sera from vaccinated volunteers have subtype-specific neutralizing antibodies against primary HIV-1 isolates. We recommend using assays with extended incubation phases to monitor current HIV vaccine efficacy trials.

Epitopes corresponding to the envelope genetic subtype are present on the surface of free virions of HIV-1 group M primary isolates and can be detected in neutralization assays with extended incubation phases.

The hypothesis is that there are neutralizing epitopes on the surface of free virions of human immunodeficiency virus type 1 (HIV-1) that correspond to the genetic subtype of the envelope glycoprotein. Assays with extended incubation and reduced absorption phases are required to demonstrate neutralization with antibodies to these epitopes. These assays quantify virus infectivity, rather than reductions in release of antigen into culture supernatants. Neutralizing antibodies reduce virus infectivity by at least 80%, as scored by the presence/absence of antigen released after 14 days in culture of mitogen-transformed peripheral blood mononuclear cells (PBMCs). The epitopes are shared within different subtypes of group M, but not group O, isolates. Individual plasma, selected from three, independent panels of seropositive individuals, cross-neutralize within each subtype as well as the combinations of A with C, B with D or G, and C with CRF01_AE. Isolates within subtype B show the greatest variation in their resistance to neutralization, ranging from highly sensitive to highly resistant. No highly sensitive subtype D isolates were identified. Isolates from subtypes A, C, and CRF01_AE were all resistant. The strategic implication for vaccine design is that antibodies to a limited number of epitopes can neutralize more than 90% of the HIV-1 isolates that are circulating currently in the world. Also, since only antibodies that produce an all-or-nothing loss in virus infectivity can reasonably be expected to prevent the viremic phase after in vivo infection, assays with extended incubation, and culture phases should be used to monitor current efficacy trials.