Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen.
ABSTRACT: Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ??0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n?=?108) and HBsAg-negative (n?=?315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).
Project description:BACKGROUND:Accurate, rapid, and cost-effective screening tests for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection may be useful in laboratories that cannot afford automated chemiluminescent immunoassays (CLIAs). We evaluated the diagnostic performance of a novel rapid automated fluorescent lateral flow immunoassay (LFIA). METHODS:A fluorescent LFIA using a small bench-top fluorescence reader, Automated Fluorescent Immunoassay System (AFIAS; Boditech Med Inc., Chuncheon, Korea), was developed for qualitative detection of hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), and antibody to HCV (anti-HCV) within 20 minutes. We compared the diagnostic performance of AFIAS with that of automated CLIAs-Elecsys (Roche Diagnostics GmbH, Penzberg, Germany) and ARCHITECT (Abbott Laboratories, Abbott Park, IL, USA)-using 20 seroconversion panels and 3,500 clinical serum samples. RESULTS:Evaluation with the seroconversion panels demonstrated that AFIAS had adequate sensitivity for HBsAg and anti-HCV detection. From the clinical samples, AFIAS sensitivity and specificity were 99.8% and 99.3% for the HBsAg test, 100.0% and 100.0% for the anti-HBs test, and 98.8% and 99.1% for the anti-HCV test, respectively. Its agreement rates with the Elecsys HBsAg, anti-HBs, and anti-HCV detection assays were 99.4%, 100.0%, and 99.0%, respectively. AFIAS detected all samples with HBsAg genotypes A-F and H and anti-HCV genotypes 1, 1a, 1b, 2a, 2b, 4, and 6. Cross-reactivity with other infections was not observed. CONCLUSIONS:The AFIAS HBsAg, anti-HBs, and anti-HCV tests demonstrated diagnostic performance equivalent to current automated CLIAs. AFIAS could be used for a large-scale HBV or HCV screening in low-resource laboratories or low-to middle-income areas.
Project description:BACKGROUND:Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". METHODS:Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. RESULTS:The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. CONCLUSIONS:According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.
Project description:Early diagnosis of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is pivotal for optimal disease management. Sensitivity and specificity of 19 rapid diagnostic test (RDT) kits by different manufacturers (ABON, CTK Biotech, Cypress Diagnostics, Green Gross, Human Diagnostic, Humasis, InTec, OraSure, SD Bioline, Wondfo) were assessed on serum samples of 270 Mongolians (90 seropositive for hepatitis B surface antigen (HBsAg), 90 seropositive for hepatitis C antibody (HCV-Ab), 90 healthy subjects). All tested RDTs for detection of HBsAg performed with average sensitivities and specificities of 100% and 99%, respectively. Albeit, overall sensitivity and specificity of RDTs for detection of HCV-Ab was somewhat lower compared to that of HBsAg RDTs (average sensitivity 98.9%, average specificity 96.7%). Specificity of RDTs for detection of HCV-Ab was dramatically lower among HBsAg positive individuals, who were 10.2 times more likely to show false positive test results. The results of our prospective study demonstrate that inexpensive, easy to handle RDTs are a promising tool in effective HBV- and HCV-screening especially in resource-limited settings.
Project description:<h4>Background</h4>White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses.<h4>Methods/principal findings</h4>Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR.<h4>Conclusion</h4>The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.
Project description:BACKGROUND AND OBJECTIVES: This study was conducted by the International Consortium for Blood Safety (ICBS) to identify high-quality test kits for detection of hepatitis B virus (HBV) surface antigen (HBsAg) for the benefit of developing countries. MATERIALS AND METHODS: The 70 HBsAg test kits from around the world were evaluated comparatively for their clinical sensitivity, analytical sensitivity, sensitivity to HBV genotypes and HBsAg subtypes, and specificity using 394 (146 clinical, 48 analytical and 200 negative) ICBS Master Panel members of diverse geographical origin comprising the major HBV genotypes A-F and the HBsAg subtypes adw2,4, adr and ayw1-4. RESULTS: Seventeen HBsAg enzyme immunoassay (EIA) kits had high analytical sensitivity <0.13 IU/ml, showed 100% diagnostic sensitivity, and were even sensitive for the various HBV variants tested. An additional six test kits had high sensitivity (<0.13 IU/ml) but missed HBsAg mutants and/or showed reduced sensitivity to certain HBV genotypes. Twenty HBsAg EIA kits were in the sensitivity range of 0.13-1 IU/ml. The other eight EIAs and the 19 rapid assays had analytical sensitivities of 1 to >4 IU/ml. These assays were falsely negative for 1-4 clinical samples and 17 of these test kits showed genotype dependent sensitivity reduction. Analytical sensitivities for HBsAg of >1 IU/ml significantly reduce the length of the HBsAg positive period which renders them less reliable for detecting HBsAg in asymptomatic HBV infections. Reduced sensitivity for HBsAg with genetic diversity of HBV occurred with genotypes/subtypes D/ayw3, E/ayw4, F/adw4 and by S gene mutants. Specificity of the HBsAg assays was >or=99.5% in 57 test kits and 96.4-99.0% in the remaining test kits. CONCLUSION: Diagnostic efficacy of the evaluated HBsAg test kits differed substantially. Laboratories should therefore be aware of the analytical sensitivity for HBsAg and check for the relevant HBV variants circulating in the relevant population.
Project description:Chronic Hepatitis B Virus (HBV) infection is characterised by the persistence of hepatitis B surface antigen (HBsAg). Expanding HBV diagnosis and treatment programmes into low resource settings will require high quality but inexpensive rapid diagnostic tests (RDTs) in addition to laboratory-based enzyme immunoassays (EIAs) to detect HBsAg. The purpose of this review is to assess the clinical accuracy of available diagnostic tests to detect HBsAg to inform recommendations on testing strategies in 2017 WHO hepatitis testing guidelines.The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines using 9 databases. Two reviewers independently extracted data according to a pre-specified plan and evaluated study quality. Meta-analysis was performed. HBsAg diagnostic accuracy of rapid diagnostic tests (RDTs) was compared to enzyme immunoassay (EIA) and nucleic-acid test (NAT) reference standards. Subanalyses were performed to determine accuracy among brands, HIV-status and specimen type.Of the 40 studies that met the inclusion criteria, 33 compared RDTs and/or EIAs against EIAs and 7 against NATs as reference standards. Thirty studies assessed diagnostic accuracy of 33 brands of RDTs in 23,716 individuals from 23 countries using EIA as the reference standard. The pooled sensitivity and specificity were 90.0% (95% CI: 89.1, 90.8) and 99.5% (95% CI: 99.4, 99.5) respectively, but accuracy varied widely among brands. Accuracy did not differ significantly whether serum, plasma, venous or capillary whole blood was used. Pooled sensitivity of RDTs in 5 studies of HIV-positive persons was lower at 72.3% (95% CI: 67.9, 76.4) compared to that in HIV-negative persons, but specificity remained high. Five studies evaluated 8 EIAs against a chemiluminescence immunoassay reference standard with a pooled sensitivity and specificity of 88.9% (95% CI: 87.0, 90.6) and 98.4% (95% CI: 97.8, 98.8), respectively. Accuracy of both RDTs and EIAs using a NAT reference were generally lower, especially amongst HIV-positive cohorts.HBsAg RDTs have good sensitivity and excellent specificity compared to laboratory immunoassays as a reference standard. Sensitivity of HBsAg RDTs may be lower in HIV infected individuals.
Project description:Most malaria testing is by rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein 2 (HRP2). Recently, several RDT manufacturers have developed highly sensitive RDTs (hsRDTs), promising a limit of detection (LOD) orders of magnitude lower than conventional RDTs. To model the added utility of hsRDTs, HRP2 concentration in Angolan outpatients was measured quantitatively using an ultrasensitive bead-based assay. The distribution of HRP2 concentration was bimodal in both afebrile and febrile patients. The conventional RDT was able to detect 81% of all HRP2-positive febrile patients and 52-77% of HRP2-positive afebrile patients. The added utility of hsRDTs was estimated to be greater in afebrile patients, where an hsRDT with a LOD of 200 pg/mL would detect an additional 50-60% of HRP2-positive persons compared with a conventional RDT with a LOD of 3,000 pg/mL. In febrile patients, the hsRDT would detect an additional 10-20% of cases. Conventional RDTs already capture the vast majority of symptomatic HRP2-positive individuals, and hsRDTs would have to reach a sufficiently low LOD approaching 200 pg/mL to provide added utility in identifying HRP2-positive, asymptomatic individuals.
Project description:Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15?min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL-1, and exhibited a wide linear range (0.63-640 IU mL-1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.
Project description:Limited access to nucleic acid tests for hepatitis B virus (HBV) DNA is a significant barrier to the effective management of chronic HBV infection in resource-poor countries. Alternatively, HBV e antigen (HBeAg) may accurately indicate high viral replication. We assessed the diagnostic performance of three commercially available rapid diagnostic tests (RDTs) for HBeAg (SD Bioline, Insight and OneStep) against a quantitative chemiluminescent immunoassay (CLIA, Architect). Using stored sera from adults with chronic HBV infection, we tested RDTs in three groups in Senegal (48 HBeAg-positive, 196 HBeAg-negative, and 117 cases with high HBV DNA (≥ 106 IU/mL)) and one group in France (17 HBeAg-positive East Asians). In Senegal, the sensitivity and specificity for HBeAg detection were 29.8% and 100% for SD Bioline, 31.1% and 100% for Insight, and 42.5% and 98.4% for OneStep, respectively. The lower limits of detection of these RDTs were very high (> 2.5 log10 Paul Ehrlich Institut units/mL). Their low sensitivity was also confirmed in HBeAg-positive Asian samples (35.3-52.9%). The prevalence of HBeAg in highly viremic (≥ 106 IU/mL) Senegalese patients was low: 58.1% using CLIA and 24.5-37.5% using RDTs. Hepatitis B e antigen prevalence was similarly low in a subgroup of 28 Senegalese women of childbearing age with a high viral load (≥ 106 IU/mL). Approximately, half of highly viremic adults do not carry HBeAg in Africa, and HBeAg RDTs had remarkably poor analytical and diagnostic sensitivity. This implies that HBeAg-based antenatal screening, particularly if using the currently available HBeAg RDTs, may overlook most pregnant women at high risk of mother-to-child transmission in Africa.
Project description:BACKGROUND: Rapid point-of-care tests provide plausible diagnostic strategy for hepatitis B surface antigen (HBsAg) in low resource areas. However, their utility depends upon their overall performance. Our objective was to meta-analyze the diagnostic accuracy of rapid point-of-care tests for HBsAg. METHODS: Literature search was done with the help of a metasearch engine Mett?, a query interface for retrieving articles from five leading medical databases. Studies that employed rapid point-of-care tests for detection of HBsAg and compared the results with reference test were included. Two reviewers performed quality assessment of the studies and extracted data for estimating test accuracy. Twenty-seven studies were meta-analyzed and stratified by multiple parameters. RESULTS: Twenty-seven studies had evaluated 49 test brands and generated 76 data points. Sensitivity of individual tests varied widely and were heterogeneous (range 43.5%-99.8%); while specificity estimates were more robust and close to 100% (range 90%-100%). Overall pooled sensitivity, specificity, positive likelihood ratio (LR), negative LR and diagnostic odds ratio for all tests were 97.1% (95% CI, 96.1%-97.9%), 99.9% (CI, 99.8%-100%), 118.4 (CI, 84.7-165.5), 0.032 (CI, 0.023-0.045) and 4094.7 (CI, 2504.1-6600.8) respectively. This suggested high pooled accuracy for all studies. We found substantial heterogeneity between studies. Three factors (study location, reference standard and study score) appeared most strongly associated with test estimates and observed heterogeneity. The Determine test showed consistency in performance in studies done across developed and developing countries and the Determine and the BinaxNOW tests had significantly higher estimates than pooled estimates of remaining tests. Tests revealed analytical sensitivity of 4 IU/ml against manufacturer's claim of 0.5 IU/ml; reduced sensitivity with HBsAg mutants and poor performance in seroconversion panels. CONCLUSIONS: Tests with better analytical sensitivity need to be developed and their feasibility and outcomes in various clinical settings need to be addressed.