Detection of Recent HIV-1 Infection Using a New Limiting-Antigen Avidity Assay: Potential for HIV-1 Incidence Estimates and Avidity Maturation Studies

Background: Accurate and reliable laboratory methods are needed for estimation of HIV-1 incidence to identify the highrisk populations and target and monitor prevention efforts. We previously described a single-well limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) to detect recent HIV-1 infection. Methods: We describe here further optimization and characterization of LAg-Avidity EIA, comparing it to the BED assay and a two-well avidity-index (AI) EIA. Specimen sets included longitudinal sera (n = 393), collected from 89 seroconverting individuals from 4 cohorts representing 4 HIV-1 subtypes, and sera from AIDS patients (n = 488) with or without TB coinfections from 3 different cohorts. Ninety seven HIV-1 positive specimens were purchased commercially. The BED assay, LAg-Avidity EIA, AI-EIA and HIV serology were performed, as needed. Results: Monitoring quality control specimens indicated high reproducibility of the LAg-Avidity EIA with coefficient of variation of,10 % in the dynamic range. The LAg-Avidity EIA has an overall mean duration of recency (v) of 141 days (95% CI 119–160) at normalized optical density (ODn) cutoff of 1.0, with similar v in different HIV-1 subtypes and populations (132 to 143 days). Antibody avidity kinetics were similar among individuals and subtypes by both the LAg-Avidity EIA and AI-EIA compared to the HIV-IgG levels measured by the BED assay. The false recent rate among individuals with AIDS was 0.2% with the LAg-Avidity EIA, compared to 2.9 % with the BED assay. Western blot profiles of specimens with increasing avidit

Next generation sequencing-based investigation of potential patient-to-patient hepatitis C virus transmission during hemodialytic treatment

We investigated potential patient-to-patient transmission of hepatitis C virus (HCV) in two hemodialysis centers in Beijing, China. Approximately 8.25% (32/388) hemodialysis patients were HCV antibody positive, and 4.90% (19/388) were HCV RNA-positive, which consisted of 2a genotype (1/19) and 1b genotypes (18/19). Using next generation sequencing (NGS) approach, MiSeq platform, we sequenced HCV, targeting hypervariable region 1 (263 base-pairs) of genotype 1b specimens and obtained 18 to 243 unique HCV variants. Analysis of phylogenetic tree, viral epidemiology signature pattern (VESP) and Shannon entropy indicated no obvious HCV similarity for most HCV infections but limited HCV variants from Patient 31 (P31) were closer with respect to evolutionary relationship with Patient 24 (P24). However, it was unlikely that HCV was transmitted directly from P24 to P31 in the hemodialysis center. Otherwise, their genetic distance (3.92%-8.92%), would have been much less. Moreover, P31 was infected less than two years before specimen collection, and other external high risk factors existed for these two patients. Thus, our data indicated no evidence of patient-to-patient transmission of HCV in the two hemodialysis centers, suggesting that current HCV infection control measures are effective

Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus

Antibody detection with a recombinant COOH portion of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (N) protein, N13 (amino acids 221 to 422), was demonstrated to be more specific and sensitive than that with the full-length N protein, and an N13-based antigen-capturing enzyme-linked immunosorbent assay providing a convenient and specific test for serodiagnosis and epidemiological study of SARS was developed

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

Abstract Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.</p

Serum angiopoietin-2 and β-hCG as predictors of prolonged uterine bleeding after medical abortion in the first trimester.

OBJECTIVE: The combination of mifepristone and misoprostol is an established method for induction of early first trimester abortion, but there is no consensus about the best evaluation of treatment outcome. We evaluate serum Angiopoietin-2 (Ang-2) and β human chorionic gonadotropin (β-hCG) in women who had undergone a medical abortion as markers of prolonged uterine bleeding (PUB). METHODS: Prospective trial involving 2843 women attending an gynecology outpatient clinic who following a medical abortion with mifepristone and misoprostol, the study cohort was divided into women with duration of uterine bleeding >14 days (PUB) and women with duration of uterine bleeding ≤14 days (normal uterine bleeding, NUB). Serum determinations of Ang-2 levels by ELISA and β-hCG levels by electrochemiluminiscence immunoassay. Receiver Operating Characteristics (ROC) analyses were calculated and plotted for the diagnostic accuracy of serum β-hCG and Ang-2 concentration to discriminate PUB and NUB. RESULTS: Baseline characteristics for both groups were similar, Only duration of bleeding showed a significant difference between the PUB group and NUB group. Ang-2 serum levels moderately correlated with serum β-hCG levels with statistically significant correlation coefficients of 0.536. Serum β-hCG and Ang-2 levels on day 7 and on day 14 after medical abortion were signifcantly higher in PUB group than in NUB group. Plotted as ROC curves, β-hCG area under curve (AUC) was 0.65 (95% CI, 0.53-0.76) on day 7, rising to AUC = 0.83 (95% CI, 0.75-0.92) on day 14. Using Ang-2 on day 7 and day 14 as predictive parameter resulted in an analogous AUC (AUC = 0.61 on day 7, AUC = 0.78 on day 14). CONCLUSIONS: Both parameters are clinically useful as a diagnostic test in predicting PUB after medical abortion, and can be helpful in uncertain clinical situations, but should be considered as supplementary to a general clinical evaluation

METHODOLOGY Open Access Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

Background: HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. Method: A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results: The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded a

Development of a New Limiting-Antigen Avidity Dot Immuno-Gold Filtration Assay for HIV-1 Incidence

<div><p>Several laboratory assays on cross-sectional specimens for detecting recent HIV infections were developed, but these assays could not be applied in resource-limited and high HIV-incidence areas. This study describes the development of a rapid assay that can simultaneously detect the presence of HIV-1 antibodies of current and/or recent infection. The dot immuno-gold filtration assay (DIGFA) was used to detect recent infection on the principle of antibody avidity changes between recent and long-term infections. The dot immuno-gold silver staining filtration assay (DIGSSA) increases the sensitivity and accuracy of antibody detection by adding a silver staining step to the DIGFA. In the meantime the digital results were produced by the scanner for ambiguous specimens. Further, HIV-1 routine diagnostic antibody was detected simultaneously for improving practicability. The performance of the assays was then assessed through five serum panels with known serological statuses and seroconversion dates. The proportion of false recent infection (PFR) of the DIGSSA was obtained. Through the optimization of basic parameters for DIGSSA, six specimens were all classified correctly. DIGSSA demonstrated good repeatability and high sensitivity. The agreement of DIGSSA with the BED assay was 92.10% (κ = 0.65) and 95.36% with the LAg-Avidity assay (κ = 0.75). Moreover, the gray values of DIGSSA correlated well with BED ODn (R² = 0.9397) and LAg-Avidity ODn (R² = 0.9549). The PFR of DIGSSA was 2.73%, which was lower than that of the BED assay but higher than that of the LAg-Avidity assay. The DIGSSA can feasibly be applied to detect HIV infection and estimate HIV incidence.</p></div