A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus.

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102-105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Independent Evaluation of Two Prototype Immunochromatographic Tests for Dengue Fever Developed by InBios.

INTRODUCTION: Dengue fever, caused by any of the four dengue viruses (DENV1-4), is endemic in more than 100 countries around the world. Each year, up to 400 million people get infected with dengue virus. It is one of the most important arthropod-borne viral diseases. Dengue's global presence poses a medical threat to deploying military personnel and their dependents. An accurate diagnosis followed by attentive supportive care can improve outcomes in patients with severe dengue disease. Dengue diagnostic tests based on PCR and ELISA platforms have been developed and cleared by the U.S. FDA. However, these diagnostic assays are laborious and usually require highly trained personnel and specialized equipment, which presents a significant challenge when conducting operations in austere and resource-constrained areas. InBios International, Inc. (Seattle, WA) has developed two rapid and instrument-free immunochromatographic test prototype devices (multiplex and traditional formats) for dengue diagnosis. MATERIALS AND METHODS: To determine the performance of the InBios immunochromatographic tests, 183 clinical samples were tested on both prototype devices. Both assays were performed without any instruments and the results were read in 20 minutes. RESULTS: The traditional format had better overall performance (sensitivity: 97.4%; specificity: 90%) than the multiplex format (sensitivity: 86.9%; specificity: 63.3%). The traditional format was superior in serotype-specific detection with 100% overall sensitivity for DENV1, DENV3, and DENV4 and 93.3% sensitivity for DENV2 compared to the multiplex format (91.7%, 78.3%, 83.3%, and 96.3% for DENV1, 2, 3, and 4, respectively). The traditional format was easier to read than the multiplex format. The multiplex format was simpler and faster to set up than the traditional format. CONCLUSIONS: The InBios traditional format had a better overall performance and readability profile than the multiplex format, while the multiplex format was easier to set up. Both formats were highly sensitive and specific, were easy to perform, and did not require sophisticated equipment. They are ideal for use in resource-limited settings where dengue is endemic. Based on our overall assessment, the traditional format should be considered for further development and used in the upcoming multicenter clinical trial toward FDA clearance.

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

Human herpesvirus 8 infection among various population groups in southern Israel.

OBJECTIVE: To compare the prevalence of antibodies to human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus among Israeli and Ethiopian subjects. METHODS: Serum samples were obtained from 98 Israeli Jewish students aged 18-30 years, 100 HIV-1-seronegative Ethiopian immigrants to Israel of the same age, and 100 HIV-1-seronegative Ethiopian children 1-12 years old upon their arrival in southern Israel. Plasma samples were obtained from 3 hospitalized patients with multicentric Castleman disease (MCD) as positive controls. All serum samples were tested for antibodies to both latent and lytic antigens. Antibodies to the lytic antigens and the latency-associated nuclear antigen (LANA) of HHV-8 were detected by enzyme linked immunosorbent assay and by immunofluorescence assay. HHV-8 DNA from serum or plasma samples was detected by polymerase chain reaction analysis. RESULTS: Antibodies to HHV-8 LANA were detected in 2.9% of the Israeli subjects aged 18-30 years and in 26% of the Ethiopian subjects from both age groups tested. Antibodies to the lytic antigens were detected in all 3 MCD patients, in 4% of the Ethiopian children, and in 2% of the 18- to 30-year-old Ethiopians. No antibodies to the lytic antigens were detected in the Israeli students. HHV-8 DNA was detected in all 3 MCD patients and in 2 of 4 of the Ethiopian children positive for the lytic antigens. CONCLUSIONS: HHV-8 is highly prevalent in Ethiopian immigrants to Israel as compared with Israeli students. Antibodies to HHV-8 in Ethiopia are acquired before puberty. The results of this study indicate the association of HHV-8 with MCD, as has been documented by many other researchers.