Seon-Ju Yeo

Smartphone-Based Fluorescent Diagnostic System for Highly Pathogenic H5N1 Viruses

Field diagnostic tools for avian influenza (AI) are indispensable for the prevention and controlled management of highly pathogenic AI-related diseases. More accurate, faster and networked on-site monitoring is demanded to detect such AI viruses with high sensitivity as well as to maintain up-to-date information about their geographical transmission. In this work, we assessed the clinical and field-level performance of a smartphone-based fluorescent diagnostic device with an efficient reflective light collection module using a coumarin-derived dendrimer-based fluorescent lateral flow immunoassay. By application of an optimized bioconjugate, a smartphone-based diagnostic device had a two-fold higher detectability as compared to that of the table-top fluorescence strip reader for three different AI subtypes (H5N3, H7N1, and H9N2). Additionally, in a clinical study of H5N1-confirmed patients, the smartphone-based diagnostic device showed a sensitivity of 96.55% (28/29) [95% confidence interval (CI): 82.24 to 99.91] and a specificity of 98.55% (68/69) (95% CI: 92.19 to 99.96). The measurement results from the distributed individual smartphones were wirelessly transmitted via short messaging service and collected by a centralized database system for further information processing and data mining. Smartphone-based diagnosis provided highly sensitive measurement results for H5N1 detection within 15 minutes. Because of its high sensitivity, portability and automatic reporting feature, the proposed device will enable agile identification of patients and efficient control of AI dissemination.

Rapid and Quantitative Detection of Zoonotic Influenza A Virus Infection Utilizing Coumarin-derived dendrimer-based Fluorescent Immunochromatographic Strip Test (FICT)

Great efforts have been made to develop robust signal-generating fluorescence materials which will help in improving the rapid diagnostic test (RDT) in terms of sensitivity and quantification. In this study, we developed coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT) assay with enhanced sensitivity as a quantitative diagnostic tool in typical RDT environments. The accuracy of the proposed FICT was compared with that of dot blot immunoassay techniques and conventional RDTs. Through conjugation of coumarin-derived dendrimers with latex beads, fluorescent emission covering broad output spectral ranges was obtained which provided a distinct advantage of easy discrimination of the fluorescent emission of the latex beads with a simple insertion of a long-pass optical filter away from the excitation wavelength. The newly developed FICT assay was able to detect 100 ng/10 μL of influenza A nucleoprotein (NP) antigen within 5 minutes, which corresponded to 2.5-fold higher sensitivity than that of the dot blot immunoassay or conventional RDTs. Moreover, the FICT assay was confirmed to detect at least four avian influenza A subtypes (H5N3, H7N1, H7N7, and H9N2). On applying the FICT to the clinical swab samples infected with respiratory viruses, our FICT assay was confirmed to differentiate influenza H1N1 infection from other respiratory viral diseases. These data demonstrate that the proposed FICT assay is able to detect zoonotic influenza A viruses with a high sensitivity, and it enables the quantitation of the infection intensity by providing the numerical diagnostic values; thus demonstrating enhanced detectability of influenza A viruses.

Improvement of a rapid diagnostic application of monoclonal antibodies against avian influenza H7 subtype virus using Europium nanoparticles

The development of a sensitive and rapid diagnostic test is needed for early detection of avian influenza (AI) H7 subtype. In this study, novel monoclonal antibodies (mAbs) against influenza A H7N9 recombinant hemagglutinin (rHA)1 were developed and applied to a Europium nanoparticle–based rapid fluorescent immunochromatographic strip test (FICT) to improve the sensitivity of the rapid diagnostic system. Two antibodies (2F4 and 6D7) exhibited H7 subtype specificity in a dot-FICT assay by optimization of the conjugate and the pH of the lysis buffer. The subtype specificity was confirmed by an immunofluorescence assay and Western blot analysis. The limit of detection of the FICT employing novel mAbs 31 ng/mL for H7N9 rHA1 and 40 hemagglutination units/mL for H7 subtype virus. Sensitivity was improved 25-fold using Europium as confirmed by comparison of colloidal gold-based rapid diagnostic kit using the 2F4 and 6D7 mAbs.

Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye.

Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens.

In vitro and in vivo effects of nitrofurantoin on experimental toxoplasmosis.

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.

Potential Interaction of Plasmodium falciparum Hsp60 and Calpain.

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.

Development of a novel peptide aptamer-based immunoassay to detect Zika virus in serum and urine

Zika virus (ZIKV) has been identified as a cause of adverse outcomes of pregnancy, including microcephaly and other congenital diseases. Most people infected with ZIKV do not show any symptoms. Development of a method to discriminate dengue virus (DENV) and ZIKV infections has been challenging, and efficient assays for patient management are limited, attributable to high levels of cross-reactivity among co-circulating Flaviviruses. Thus, there is an urgent need for a specific high-throughput diagnostic assay to discriminate ZIKV infections from other Flavivirus infections. Methods: A novel epitope peptide of the ZIKV envelope protein was predicted using three immune epitope database analysis tools and then further modified. A molecular docking study was conducted using three-dimensional structures of the ZIKV envelope and peptide. Experimentally, interactions between the selected peptides and virus were assessed via a fluorescence-linked sandwich immunosorbent assay (FLISA), and performance of peptide-linked sandwich FLISA was evaluated in virus-spiked human serum and urine. Results: The Z_10.8 peptide (KRAVVSCAEA) was predicted to be a suitable detector, with a higher binding affinity than other candidates based on four criteria (binding affinity, root mean square deviation, position of amine residue of lysine at the N-terminus, and interactive site) in a docking study. Z_10.8 was significantly more efficient at detecting ZIKV than the other two peptides, as shown in the direct FLISA (P < 0.001). Further, the equilibrium dissociation constant (Kd) for the Z_10.8 peptide was 706.0 ± 177.9 (mean ± SD, nM), with specificity to discriminate ZIKV from DENV. The limit of detection for the sandwich FLISA was calculated as 1×104 tissue culture infective dose (TCID)50/mL. The presence of serum or urine did not interfere with the performance of the Z_10.8-linked sandwich FLISA. Conclusion: Four criteria are suggested for the development of an in silico modeled peptide aptamer; this computerized peptide aptamer discriminated ZIKV from DENV via immunoassay.

Clinical evaluation of rapid fluorescent diagnostic immunochromatographic test for influenza A virus (H1N1)

Rapid diagnostic tests (RDTs) have been developed to detect influenza A virus for the swift diagnosis and management of patients. However, despite the simplicity and convenience, the low sensitivity of RDTs remains a limitation for their use in point of care testing (POCT). In this study, we developed a rapid fluorescent immunochromatographic strip test (FICT) and the performance of FICT was confirmed by the real-time reverse transcription-polymerase chain reaction (rRT-PCR) of H1N1, compared with that of RDT. The limit of detection (LOD) of FICT was improved by 16-fold compared to RDT. FICT showed 85.29% sensitivity (29/34) (95% Confidence Interval [95% CI]: 68.94 to 95.05), 100% specificity (26/26) (95% CI: 86.78 to 100.00), and a strong correlation (kappa; 0. 92) compared with rRT-PCR (20 ≤ Ct ≤ 36). In contrast, RDT (Standard Diagnostics [SD] BIOLINE Influenza Ag A/ B/ A(H1N1) Pandemic) showed 55.88% sensitivity (19/34) (95% CI: 37.87 to 72.82), 100% specificity (26/26) (95% CI: 77.07 to 100), and had a fair correlation with rRT-PCR (kappa; 0. 75). FICT had better sensitivity than RDT (P < 0.01; McNemar’s test). Therefore, FICT has the potential to improve the quality of current rapid POCT for the diagnosis of influenza A/H1N1 infection.

Development of a Rapid Fluorescent Immunochromatographic Test to Detect Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is one of the most common viruses infecting the respiratory tracts of infants. The rapid and sensitive detection of RSV is important to minimize the incidence of infection. In this study, novel monoclonal antibodies (mAbs; B11A5 and E8A11) against RSV nucleoprotein (NP) were developed and applied to develop a rapid fluorescent immunochromatographic strip test (FICT), employing europium nanoparticles as the fluorescent material. For the FICT, the limits of detection of the antigen and virus were 1.25 µg/mL and 4.23 × 106 TCID50/mL, respectively, corresponding to 4.75 × 106 ± 5.8 ×105 (mean ± SD) RNA copy numbers per reaction mixture for RSV NP. A clinical study revealed a sensitivity of 90% (18/20) and specificity of 98.18% (108/110) for RSV detection when comparing the performance to that of reverse transcription polymerase chain reaction (RT-PCR), representing a 15% improvement in sensitivity over the SD Bioline rapid kit. This newly developed FICT could be a useful tool for the rapid diagnosis of RSV infection.

Rapid Detection of Avian Influenza Virus by Fluorescent Diagnostic Assay using an Epitope-Derived Peptide

Currently, the point of care testing (POCT) is not fully developed for subtype-specific avian influenza virus detection. In this study, an H5N1 hemaglutinin 1 (HA1) epitope (P0: KPNDAINF) and three modified peptides (P1: KPNTAINF, P2: KPNGAINF, P3: KPNDAINDAINF) were evaluated as POCT elements for rapid detection of avian influenza virus. Based on modeling predictions by Autodock Vina, binding affinity varied depending on alteration of one amino acid in these peptides. The binding energy of P2 indicated its potential for a strong interaction with HA. Fluorescence-linked immunosorbent assay experimentally demonstrated the interaction between these peptides and virus. The four peptides interacted with HA1 of H5N3 with different binding affinities with P2 showing the strongest binding affinity. When P0 and P2 peptides were used in rapid fluorescent immunochromatographic test (FICT) as detection elements, the inter-assay coefficients of variation (CV) indicated that P2-linked FICT was more acceptable than the P0-linked FICT in the presence of human specimens. Antibody pair-linked FICT was influenced by clinical samples more than the P2-linked FICT assay, which showed a 4-fold improvement in the detection limit of H5N3 and maintained H5 subtype-specificity. Compared to the rapid diagnostic test (RDT) which is not specific for influenza subtypes, P2-linked FICT could increase virus detection. In conclusion, results of this study suggest that HA epitope-derived peptides can be used as alternatives to antibodies for a rapid fluorescent diagnostic assay to detect avian influenza virus.