Evaluation of a smartphone-based rapid fluorescent diagnostic system for H9N2 virus in specific-pathogen-free chickens.

Repeated interspecies transmission of H9N2 virus from poultry to humans and human infections transmitted via aerosols highlight the need for a highly sensitive, rapid diagnostic system for the detection of this virus. However, no such test exhibiting high performance has been developed. In this study, the performance of a smartphone-based rapid fluorescent diagnostic system (SRFDS) was optimized for the diagnosis of an H9N2-virus-infected animal. To suppress the nonspecific reactivity of the bioconjugate in oropharyngeal (OP) and cloacal (CL) samples derived from chickens, different blocking reagents were tested, and a mixture of casein and sucrose was found to be optimal. To assess the performance of SRFDS, OP and CL samples were obtained from specific-pathogen-free chickens and used for comparison of this method with real-time reverse transcription PCR (rRT-PCR) at time points of three, five, and seven days postinfection (dpi). The limit of detection of SRFDS was found to be 7.5 PFU/mL, which was 138-fold higher than that of a conventional colloidal-gold-based avian influenza rapid diagnostic test. In the animal study, the presence of viral antigen was monitored with SRFDS, and the relative sensitivity (relative to rRT-PCR results) was 94.44 % (17/18) and 95.23 % (20/21) in OP and CL specimens, respectively. The specificity of SRFDS was 100 %. These results imply that the diagnostic performance of SRFDS might be comparable to that of rRT-PCR for diagnosis of H9N2 in chickens and that this test can be used as a highly sensitive rapid diagnostic method in field studies on broiler poultry and wild birds.

Development of a smartphone-based rapid dual fluorescent diagnostic system for the simultaneous detection of influenza A and H5 subtype in avian influenza A-infected patients.

Accurate and rapid diagnosis of highly pathogenic avian influenza A H5N1 is of critical importance for the effective clinical management of patients. Here, we developed a rapid and simultaneous detection toolkit for influenza A H5 subtype viruses in human samples based on a bioconjugate of quantum dots (QDs) assembly and a smartphone-based rapid dual fluorescent diagnostic system (SRDFDS). Methods: Two types of QDs were assembled on a latex bead to enhance the detection sensitivity and specificity of influenza A infection (QD580) and H5 subtype (QD650). The dual signals of influenza A and H5 subtype of H5N1-infected patients were detected simultaneously and quantified separately by SRDFDS equipped with two emission filters. Results: Our results showed a high sensitivity of 92.86% (13/14) and 78.57% (11/14), and a specificity of 100% (38/38, P < 0.0001) and 97.37% (37/38) for influenza A and H5 subtype detection, respectively. Conclusion: Therefore, our multiplex QD bioconjugates and SRDFDS-based influenza virus detection toolkit potentially provide accurate and meaningful diagnosis information with improved detection accuracies and sensitivities for H5N1 patients.

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.

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.

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.