High level of HIV false positives using EIA-based algorithm in survey: Importance of confirmatory testing.

The Mozambique Indicators of Immunization, Malaria and HIV/AIDS (IMASIDA) survey was conducted in 2015 and used a two Enzyme Immunoassay (EIA) (Vironostika HIV-1/2 and Murex HIV-1/2) based algorithm to determine the HIV status of the consented participants. The Mozambique Ministry of Health, with support from the US Centers for Disease Control and Prevention (US CDC), added Bio-Rad Geenius™ HIV-1/2 Supplemental Assay to the IMASIDA HIV testing algorithm to confirm all specimens that were found to be reactive on one or both EIAs. In total 11690 specimens were collected to estimate the proportion of HIV positive samples. Results indicate that the proportion of HIV positive samples based on the concordant positive results of two EIA assays was 21.5% (2518/11690). The addition of the Geenius assay to the IMASIDA HIV testing algorithm demonstrated that 792 (31.5%) of 2518 specimens were false-positive and reduced the proportion of HIV positive samples to 14.7% (1722/11690), demonstrating the importance of including a highly specific HIV test to confirm HIV diagnosis. HIV surveys exclusively based on EIA testing algorithm may result in misleading high prevalence results. Our results demonstrate that more specific confirmatory testing should be added to the EIA-based algorithms to ensure accurate HIV diagnosis and correct HIV prevalence estimate in cross-sectional surveys.

Onsite healthcare worker acceptability and performance of the point-of-care Pima CD4 assay in Dar es Salaam, Tanzania.

BACKGROUND: Healthcare workers' acceptance of and ability to perform point-of-care testing is important for reliable and accurate results. The Alere Pima™ CD4 assay (Pima CD4) is the CD4 point-of-care test for HIV management in Tanzania. OBJECTIVES: To evaluate healthcare workers' acceptance and performance of Pima CD4 testing. METHODS: The study was implemented in five high volume sites in Dar es Salaam, Tanzania, in 2011. Trained healthcare workers performed Pima testing using three whole-blood specimens collected from each patient: venous blood, fingerstick blood directly applied to a Pima cartridge (capillary-direct), and fingerstick blood collected in a microtube (capillary-microtube). Using a semi-structured interview guide, we interviewed 11 healthcare workers about specimen collection methods and Pima CD4 acceptability. Quantitative responses were analysed using descriptive statistics. Open-ended responses were summarised by thematic areas. Pima CD4 results were analysed to determine variation between cadres. RESULTS: Healthcare workers found Pima CD4 user-friendly and recommended its use in low volume, peripheral facilities. Both venous and capillary-direct blood were considered easy to collect, with venous preferred. Advantages noted with venous and capillary-microtube methods were the ability to retest, perform multiple tests, or delay testing. Pima CD4 results were trusted by the healthcare workers and were in agreement with laboratory Pima testing. CONCLUSION: In this point-of-care testing setting, the Pima CD4 assay was accepted by healthcare workers. Both venous and fingerstick capillary blood specimens can be used with Pima CD4, but fingerstick methods may require more intensive training on technique to minimise variation in results and increase acceptability.

Low and Decreasing Prevalence and Rate of False Positive HIV Diagnosis - Chókwè District, Mozambique, 2014-2017.

In 2017, rapid human immunodeficiency virus (HIV) testing services enabled the HIV diagnosis and treatment of approximately 15.3 million persons with HIV infection in sub-Saharan Africa with life-saving antiretroviral therapy (ART) (1). Although suboptimal testing practices and misdiagnoses have been reported in sub-Saharan Africa and elsewhere, trends in population burden and rate of false positive HIV diagnosis (false diagnosis) have not been reported (2,3). Understanding the population prevalence and trends of false diagnosis is fundamental for guiding rapid HIV testing policies and practices. To help address this need, CDC analyzed data from 57,655 residents aged 15-59 years in the Chókwè Health and Demographic Surveillance System (CHDSS) in Mozambique to evaluate trends in the rate (the percentage of false diagnoses among retested persons reporting a prior HIV diagnosis) and population prevalence of false diagnosis. From 2014 to 2017, the observed rate of false diagnosis in CHDSS decreased from 0.66% to 0.00% (p<0.001), and the estimated population prevalence of false diagnosis decreased from 0.08% to 0.01% (p = 0.0016). Although the prevalence and rate of false diagnosis are low and have decreased significantly in CHDSS, observed false diagnoses underscore the importance of routine HIV retesting before ART initiation and implementation of comprehensive rapid HIV test quality management systems (2,4,5).

Evaluation of Specimen Types for Pima CD4 Point-of-Care Testing: Advantages of Fingerstick Blood Collection into an EDTA Microtube.

INTRODUCTION: Effective point-of-care testing (POCT) is reliant on optimal specimen collection, quality assured testing, and expedited return of results. Many of the POCT are designed to be used with fingerstick capillary blood to simplify the blood collection burden. However, fingerstick blood collection has inherent errors in sampling. An evaluation of the use of capillary and venous blood with CD4 POCT was conducted. METHODS: Three different specimen collection methods were evaluated for compatibility using the Alere Pima CD4 assay at 5 HIV/AIDS healthcare sites in Dar es Salaam, Tanzania. At each site, whole blood specimens were collected from enrolled patients by venipuncture and fingerstick. Pima CD4 testing was performed at site of collection on venipuncture specimens (Venous) and fingerstick blood directly applied to a Pima CD4 cartridge (Capillary-Direct) and collected into an EDTA microtube (Capillary-Microtube). Venous blood was also tested at the laboratory by the reference CD4 method and Pima for comparison analysis. RESULTS: All three specimen collection methods were successfully collected by healthcare workers for use with the Pima CD4 assay. When compared to the reference CD4 method, Pima CD4 testing with the Capillary-Microtube method performed similarly to Venous, while Pima CD4 counts with the Capillary-Direct method were slightly more biased (-20 cells/µL) and variable (-229 to +189 cells/µL limit of agreement). Even though all three collection methods had similar invalid Pima testing rates (10.5%, 9.8%, and 8.3% for Capillary-Direct, Capillary-Microtube, and Venous respectively), the ability to perform repeat testing with Capillary-Microtube and Venous specimens increased the likelihood of acquiring a valid CD4 result with the Pima assay. CONCLUSIONS: Capillary blood, either directly applied to Pima CD4 cartridges or collected in an EDTA microtube, and venous blood are suitable specimens for Pima CD4 testing. The advantages of capillary blood collection in an EDTA microtube are that it uses fingerstick collection which mimics venous blood and allows extra testing without additional blood collection.

A quality management systems approach for CD4 testing in resource-poor settings.

Quality assurance (QA) is a systematic process to monitor and improve clinical laboratory practices. The fundamental components of a laboratory QA program include providing a functional and safe laboratory environment, trained and competent personnel, maintained equipment, adequate supplies and reagents, testing of appropriate specimens, internal monitoring of quality, accurate reporting, and external quality assessments. These components are necessary to provide accurate and precise CD4 T-cell counts, an essential test to evaluate start of and monitor effectiveness of antiretroviral therapy for HIV-infected patients. In recent years, CD4 testing has expanded dramatically in resource-limited settings. Information on a CD4 QA program as described in this article will provide guidelines not only for clinical laboratory staff but also for managers of programs responsible for supporting CD4 testing. All agencies involved in implementing CD4 testing must understand the needs of the laboratory and provide advocacy, guidance, and financial support to established CD4 testing sites and programs. This article describes and explains the procedures that must be put in place to provide reliable CD4 determinations in a variety of settings.

Galectin-3 induces death of Candida species expressing specific beta-1,2-linked mannans.

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear beta-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks beta-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific beta-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry.

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.

Novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by Nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines.

Galectin-1 (gal-1), an endogenous lectin secreted by a variety of cell types, has pleiotropic immunomodulatory functions, including regulation of lymphocyte survival and cytokine secretion in autoimmune, transplant disease, and parasitic infection models. However, the role of gal-1 in viral infections is unknown. Nipah virus (NiV) is an emerging pathogen that causes severe, often fatal, febrile encephalitis. The primary targets of NiV are endothelial cells. NiV infection of endothelial cells results in cell-cell fusion and syncytia formation triggered by the fusion (F) and attachment (G) envelope glycoproteins of NiV that bear glycan structures recognized by gal-1. In the present study, we report that NiV envelope-mediated cell-cell fusion is blocked by gal-1. This inhibition is specific to the Paramyxoviridae family because gal-1 did not inhibit fusion triggered by envelope glycoproteins of other viruses, including two retroviruses and a pox virus, but inhibited fusion triggered by envelope glycoproteins of the related Hendra virus and another paramyxovirus. The physiologic dimeric form of gal-1 is required for fusion inhibition because a monomeric gal-1 mutant had no inhibitory effect on cell fusion. gal-1 binds to specific N-glycans on NiV glycoproteins and aberrantly oligomerizes NiV-F and NiV-G, indicating a mechanism for fusion inhibition. gal-1 also increases dendritic cell production of proinflammatory cytokines such as IL-6, known to be protective in the setting of other viral diseases such as Ebola infections. Thus, gal-1 may have direct antiviral effects and may also augment the innate immune response against this emerging pathogen.