ABSTRACT
Diagnostic assays that can simultaneously determine the presence of infection with multiple pathogens are key for diagnosis and surveillance. Current multiplex diagnostic assays are complex and often have limited availability. We developed a simple, multianalyte, pathogen detection assay for screening and serosurveillance using the Luminex Magpix platform that is high throughput and can be helpful in monitoring multiple diseases. The Luminex bead-based 10-plex immunoassay for the detection of HIV-1, HIV-2, Treponema pallidum, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), and HSV-2 infections was accomplished by coupling beads with specific antigens to detect IgG antibodies in plasma or serum samples. Each coupled antigen was systematically optimized, and the performance was evaluated using a panel of well-characterized specimens (n = 417) that contained antibodies to HIV-1, HIV-2, T. pallidum, HBV, HCV, HSV-1, and HSV-2. The multiplex assay had a sensitivity of 92.2% (95% Clopper-Pearson confidence interval [CI], 90.2 to 94.0%) and a specificity of 98.1% (95% CI, 97.6 to 98.7%). The sensitivities and specificities for disease-specific biomarker detection ranged from 68.7 to 100% and 95.6 to 100%, respectively. The results showed that the 10-plex immunoassay had an overall agreement of 96.7% (95% CI, 96.7 to 97.3%) with reference tests and a corresponding kappa value of 0.91 (95% CI, 0.90 to 0.93). Kappa values for the individual pathogens ranged from 0.69 to 1.00. The assay is robust and allows the simultaneous detection of antibodies to multiple antigens using a small sample volume in a high-throughput format. This assay has the potential to simplify disease surveillance by providing an alternative to expensive and highly specialized individual tests.
Subject(s)
HIV Infections , HIV-1 , Hepatitis C , Herpes Simplex , Syphilis , HIV-2 , Hepacivirus , Hepatitis B virus , Hepatitis C/diagnosis , Herpes Simplex/diagnosis , Humans , Sensitivity and Specificity , Syphilis/diagnosis , Syphilis/epidemiology , Treponema pallidumABSTRACT
Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a ß-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.
Subject(s)
Capsid/chemistry , Capsid/ultrastructure , HIV-1/chemistry , HIV-1/ultrastructure , Molecular Dynamics Simulation , Capsid Proteins/chemistry , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , Human Immunodeficiency Virus Proteins/chemistry , Human Immunodeficiency Virus Proteins/ultrastructure , Hydrophobic and Hydrophilic Interactions , Protein Multimerization , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The Sedia Biosciences Asanté rapid test for recent infection (RTRI) can identify HIV infections and characterize HIV-1 as recent or long-term infection via the positive verification (V) line and long-term line (LT) line, respectively. Tracking with Recency Assays to Control the Epidemic (TRACE) program uses RTRI assays. Successful implementation of TRACE requires high-quality test performance. The goal of this study is to evaluate the additional quality practices established for new kit lots prior to field use. Asanté lot quality control data from the manufacturer is reviewed by the Centers for Disease Control and Prevention International Laboratory Branch (CDC-ILB) in the Division of Global HIV and TB using. If a lot passes manufacturer quality control and CDC-ILB review, test kits are sent to CDC-ILB for further evaluation. Evaluation by CDC includes inter-rater reliability and linear regressions comparing the V and LT lines against reference data as well as V and LT line data between testers. A Bland-Altman analysis was conducted to assess bias and systematic error. Overall, CDC-ILB passed 29 (91%) out of 32 Sedia Biosciences Asanté kit lots that initially passed manufacturing quality control from July 2017 to May 2020. Regression analyses demonstrate that test kits are performing as expected with consistent R2≥0.92 for both V and LT lines. On average, inter-rater reliability kappa was 0.9, indicating a strong level of agreement. Bland-Altman analyses demonstrate high agreement with little to no systematic error and bias. Ongoing evaluation of new RTRI kit lots is important to ensure high quality test performance. Rejecting 9% of kit lots highlight the importance of continuing to work with manufacturers to ensure consistent kit production and quality assurance (QA) activities. Investing in effective QA measures, conducting both pre- and post-market performance data reviews, could help improve RTRI accuracy and outcomes in similar testing programs.
ABSTRACT
BACKGROUND: Lentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits. RESULTS: We provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells. CONCLUSIONS: Our results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability.
Subject(s)
HIV Core Protein p24/metabolism , HIV-1/physiology , Protein Interaction Domains and Motifs , Virus Assembly , Cell Line , DNA Mutational Analysis , HIV Core Protein p24/genetics , HIV-1/genetics , Humans , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings.
Subject(s)
HIV Infections , Trehalose , Humans , Reproducibility of Results , Quality Control , Desiccation , HIV Infections/diagnosisABSTRACT
[This corrects the article DOI: 10.1371/journal.pgph.0000316.].
ABSTRACT
We previously described development of a rapid test for recent infection (RTRI) that can diagnose HIV infection and detect HIV-1 recent infections in a single device. This technology was transferred to a commercial partner as Asante Rapid Recency Assay (ARRA). We evaluated performance of the ARRA kits in the laboratory using a well-characterized panel of specimens. The plasma specimen panel (N = 1500) included HIV-1 (N = 570), HIV-2 (N = 10), and HIV-negatives (N = 920) representing multiple subtypes and geographic locations. Reference diagnostic data were generated using the Bio-Rad HIV-1-2-O EIA/Western blot algorithm with further serotyping performed using the Multispot HIV-1/2 assay. The LAg-Avidity EIA was used to generate reference data on recent and long-term infection for HIV-1 positive specimens at a normalized optical density (ODn) cutoff of 2.0 corresponding to a mean duration of about 6 months. All specimens were tested with ARRA according to the manufacturer's recommendations. Test strips were also read for line intensities using a reader and results were correlated with visual interpretation. ARRA's positive verification line (PVL) correctly classified 575 of 580 HIV-positive and 910 of 920 negative specimens resulting in a sensitivity of 99.1% (95% CI: 98.0-99.6) and specificity of 98.9% (95% CI: 98.1-99.4), respectively. The reader-based classification was similar for PVL with sensitivity of 99.3% (576/580) and specificity of 98.8% (909/920). ARRA's long-term line (LTL) classified 109 of 565 HIV-1 specimens as recent and 456 as long-term compared to 98 as recent and 467 as long-term (LT) by LAg-Avidity EIA (cutoff ODn = 2.0), suggesting a mean duration of recent infection (MDRI) close to 6 months. Agreement of ARRA with LAg recent cases was 81.6% (80/98) and LT cases was 93.8% (438/467), with an overall agreement of 91.7% (kappa = 0.72). The reader (cutoff 2.9) classified 109/566 specimens as recent infections compared to 99 by the LAg-Avidity EIA for recency agreement of 81.8% (81/99), LT agreement of 9% (439/467) with overall agreement of 91.9% (kappa = 0.72). The agreement between visual interpretation and strip reader was 99.9% (95% CI: 99.6-99.9) for the PVL and 98.1% (95% CI: 96.6-98.9) for the LTL. ARRA performed well with HIV diagnostic sensitivity >99% and specificity >98%. Its ability to identify recent infections is comparable to the LA-Avidity EIA corresponding to an MDRI of about 6 months. This point-of-care assay has implications for real-time surveillance of new infections among newly diagnosed individuals for targeted prevention and interrupting ongoing transmission thus accelerating epidemic control.
ABSTRACT
Human Immunodeficiency Virus (HIV) diagnosis remains the gateway to HIV care and treatment. However, due to changes in HIV prevalence and testing coverage across different geopolitical zones, it is crucial to evaluate the national HIV testing algorithm as false positivity due to low prevalence could be detrimental to both the client and the service delivery. Therefore, we evaluated the performance of the national HIV rapid testing algorithm using specimens collected from multiple HIV testing services (HTS) sites and compared the results from different HIV prevalence levels across the six geopolitical zones of Nigeria. The evaluation employed a dual approach, retrospective, and prospective. The retrospective evaluation focused on a desktop review of program data (n = 492,880) collated from patients attending routine HTS from six geopolitical zones of Nigeria between January 2017 and December 2019. The prospective component utilized samples (n = 2,895) collected from the field at the HTS and tested using the current national serial HIV rapid testing algorithm. These samples were transported to the National Reference Laboratory (NRL), Abuja, and were re-tested using the national HIV rapid testing algorithm and HIV-1/2 supplementary assays (Geenius to confirm positives and resolve discordance and multiplex assay). The retrospective component of the study revealed that the overall proportion of HIV positives, based on the selected areas, was 5.7% (28,319/492,880) within the study period, and the discordant rate between tests 1 and 2 was 1.1%. The prospective component of the study indicated no significant differences between the test performed at the field using the national HIV rapid testing algorithm and the re-testing performed at the NRL. The comparison between the test performed at the field using the national HIV rapid testing algorithm and Geenius HIV-1/2 supplementary assay showed an agreement rate of 95.2%, while that of the NRL was 99.3%. In addition, the comparison of the field results with HIV multiplex assay indicated a sensitivity of 96.6%, the specificity of 98.2%, PPV of 97.0%, and Kappa Statistic of 0.95, and that of the NRL with HIV multiplex assay was 99.2%, 99.4%, 99.0%, and 0.99, respectively. Results show that the Nigeria national serial HIV rapid testing algorithm performed very well across the target settings. However, the algorithm's performance in the field was lower than the performance outcomes under a controlled environment in the NRL. There is a need to target testers in the field for routine continuous quality improvement implementation, including refresher trainings as necessary.
ABSTRACT
BACKGROUND: Conducting HIV surveys in resource-limited settings is challenging because of logistics, limited availability of trained personnel, and complexity of testing. We described the procedures and systems deemed critical to ensure high-quality laboratory data in the population-based HIV impact assessments and large-scale household surveys. METHODS: Laboratory professionals were engaged in every stage of the surveys, including protocol development, site assessments, procurement, training, quality assurance, monitoring, analysis, and reporting writing. A tiered network of household, satellite laboratories, and central laboratories, accompanied with trainings, optimized process for blood specimen collection, storage, transport, and real-time monitoring of specimen quality, and test results at each level proved critical in maintaining specimen integrity and high-quality testing. A plausibility review of aggregate merged data was conducted to confirm associations between key variables as a final quality check for quality of laboratory results. RESULTS: Overall, we conducted a hands-on training for 3355 survey staff across 13 surveys, with 160-387 personnel trained per survey on biomarker processes. Extensive training and monitoring demonstrated that overall, 99% of specimens had adequate volume and 99.8% had no hemolysis, indicating high quality. We implemented quality control and proficiency testing for testing, resolved discrepancies, verified >300 Pima CD4 instruments, and monitored user errors. Aggregate data review for plausibility further confirmed the high quality of testing. CONCLUSIONS: Ongoing engagement of laboratory personnel to oversee processes at all levels of the surveys is critical for successful national surveys. High-quality population-based HIV impact assessments laboratory data ensured reliable results and demonstrated the impact of HIV programs in 13 countries.
Subject(s)
HIV Infections/diagnosis , HIV Infections/epidemiology , HIV-1 , Laboratory Proficiency Testing/standards , Developing Countries , Epidemiological Monitoring , Health Surveys , Humans , Laboratory Personnel/education , Laboratory Personnel/standards , Quality ControlABSTRACT
BACKGROUND: HIV-1 incidence calculation currently includes recency classification by HIV-1 incidence assay and unsuppressed viral load (VL ≥ 1000 copies/mL) in a recent infection testing algorithm (RITA). However, persons with recent classification not virally suppressed and taking antiretroviral (ARV) medication may be misclassified. SETTING: We used data from 13 African household surveys to describe the impact of an ARV-adjusted RITA on HIV-1 incidence estimates. METHODS: HIV-seropositive samples were tested for recency using the HIV-1 Limiting Antigen (LAg)-Avidity enzyme immunoassay, HIV-1 viral load, ARVs used in each country, and ARV drug resistance. LAg-recent result was defined as normalized optical density values ≤1.5. We compared HIV-1 incidence estimates using 2 RITA: RITA1: LAg-recent + VL ≥ 1000 copies/mL and RITA2: RITA1 + undetectable ARV. We explored RITA2 with self-reported ARV use and with clinical history. RESULTS: Overall, 357 adult HIV-positive participants were classified as having recent infection with RITA1. RITA2 reclassified 55 (15.4%) persons with detectable ARV as having long-term infection. Those with detectable ARV were significantly more likely to be aware of their HIV-positive status (84% vs. 10%) and had higher levels of drug resistance (74% vs. 26%) than those without detectable ARV. RITA2 incidence was lower than RITA1 incidence (range, 0%-30% decrease), resulting in decreased estimated new infections from 390,000 to 341,000 across the 13 countries. Incidence estimates were similar using detectable or self-reported ARV (R2 > 0.995). CONCLUSIONS: Including ARV in RITA2 improved the accuracy of HIV-1 incidence estimates by removing participants with likely long-term HIV infection.
Subject(s)
Algorithms , Epidemiological Monitoring , HIV Infections/diagnosis , HIV-1 , Adolescent , Adult , Anti-HIV Agents/therapeutic use , Female , HIV Infections/drug therapy , Humans , Incidence , Male , Middle Aged , Young AdultABSTRACT
Laboratory assays that can accurately distinguish recent (occurring within the past year) from long-standing (>1 year) HIV infection are crucial for understanding HIV transmission dynamics in a population. However, often these efforts are confounded by inaccurate HIV diagnosis and the presence of HIV-2 in the population being surveyed. This study describes development of a multiplex assay that can simultaneously perform HIV diagnosis, HIV serotyping, and detection of recent HIV-1 infection in a single well. HIV diagnosis and HIV-2 serotyping were accomplished by coupling beads with an HIV-1 p24-gp41 fusion protein and HIV-2 peptide from gp36 immunodominant region, respectively. HIV-1 recent infection detection was accomplished by coupling beads with limiting amounts of multi-subtype gp41 immunodominant protein, recombinant immunodominant region, group M (rIDR-M). Assay conditions, including concentration of coupled antigens, were systematically optimized using well-characterized specimens with known HIV-status (positive or negative), HIV-2 specimens, and recent or long-term HIV-1 classification based on LAg-Avidity enzyme immunoassay (EIA) in a stepwise manner. Beads were then combined in a multiplex assay to evaluate its performance using large panel of specimens (n = 1,500) that included HIV-1 positive (n = 570, recent = 78, long-term = 492), HIV-2 positive (n = 31), and seronegative individuals (n = 899). The diagnostic component of the assay performed with high sensitivity (99.8%) and specificity (99.7%), while the HIV-2 serotyping sensitivity and specificity were 96.7% and 100%, respectively. There was a high correlation (R = 0.84) between the LAg-Avidity EIA and the multiplex assay for recent infection detection. The assay showed high inter- and intra-assay reproducibility with %coefficient of variation of <10% in the dynamic range. The multiplex assay has the ability to diagnose HIV infection, perform serotyping, and detect and distinguish recent from long-term HIV infections, all in a single well. This novel assay has the potential to simplify HIV surveillance by reducing the multiple steps that are otherwise required.
Subject(s)
HIV Infections/diagnosis , HIV-1/isolation & purification , HIV-2/isolation & purification , HIV Infections/virology , Humans , Sensitivity and SpecificityABSTRACT
HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.