Serologic assays for the detection and strain identification of Pteropine orthoreovirus.

Pteropine orthoreovirus (PRV), potentially of bat origin, is reported to be a causative agent of emerging respiratory tract infections among humans in Southeast Asia. We evaluated the efficacy of serologic assays using the major outer capsid and cell attachment proteins (CAP) of PRV strains in the screening, confirmation and identification of three groups of human PRV infections; Indonesian/Japanese, Indonesian/Hong Kong and Malaysian strains. The different serologic assays were tested using rabbit polyclonal antisera raised against these proteins of selected PRV strains, and validation was carried out using sera from a Miyazaki-Bali/2007 PRV-infected patient and the patient's contacts. The results of this study showed that rabbit polyclonal antisera raised against the CAP of the Miyazaki-Bali/2007 PRV strain showed the highest reactivity to the Miyazaki-Bali/2007 PRV and to a lesser extent, cross-reactivity with the HK23629/07 and Melaka PRVs, respectively. Neutralization activity against the Miyazaki-Bali/2007 PRV was observed using rabbit anti-Miyazaki-Bali/2007 PRV CAP (320) but not with rabbit anti-HK23629/07 (<20) and Melaka (<20) PRV CAP. This lack of cross-neutralization, suggests the potential for human reinfection with different strains. The use of sera collected from contacts of the Miyazaki-Bali/2007 PRV-infected patient suggested that human-to-human infections with PRV are unlikely. Previously reported cases of PRV infections among human have been mild. However, the expanding geographic distribution of these viruses, of which its virulence remains unknown, warrants close monitoring to enable the development of prevention and control strategies in the event that a change in virulence occurs.

A Handy Field-Portable ELISA System for Rapid Onsite Diagnosis of Infectious Diseases.

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of various immunological reactions and can be used for the detection of infectious diseases during outbreaks or in the care of individual patients. To be useful in the timely implementation of prevention and control measures against infectious diseases, a diagnostic modality should be rapid, accurate, and affordable. In the current study, we demonstrate the efficiency (90% less time and volume consumption compared with those of a standard 96-well ELISA), detection capability, and ease of operation of a field-portable, battery-operated ELISA system, approximately the size of a cellular phone (12 × 6 × 5.5 cm), in the serological diagnosis of measles and rubella viruses that has the potential for onsite testing such as during disease outbreaks.

High sensitivity, high surface area Enzyme-linked Immunosorbent Assay (ELISA).

BACKGROUND: Enzyme-linked immunosorbent assays (ELISA) are considered the gold standard in the demonstration of various immunological reactions with an application in the detection of infectious diseases such as during outbreaks or in patient care. OBJECTIVE: This study aimed to produce an ELISA-based diagnostic with an increased sensitivity of detection compared to the standard 96-well method in the immunologic diagnosis of infectious diseases. METHODS: A '3DStack' was developed using readily available, low cost fabrication technologies namely nanoimprinting and press stamping with an increased surface area of 4 to 6 times more compared to 96-well plates. This was achieved by stacking multiple nanoimprinted polymer sheets. The flow of analytes between the sheets was enhanced by rotating the 3DStack and confirmed by Finite-Element (FE) simulation. An Immunoglobulin G (IgG) ELISA for the detection of antibodies in human serum raised against Rubella virus was performed for validation. RESULTS: An improved sensitivity of up to 1.9 folds higher was observed using the 3DStack compared to the standard method. CONCLUSIONS: The increased surface area of the 3DStack developed using nanoimprinting and press stamping technologies, and the flow pattern between sheets generated by rotating the 3DStack were potential contributors to a more sensitive ELISA-based diagnostic device.

Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation - next generation sequencing.

The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method "modified rolling circular amplification with adaptor ligation - next generation sequencing (mRCA-NGS)". Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV.

Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

Serological evidence of human infection with Pteropine orthoreovirus in Central Vietnam.

Pteropine orthoreovirus, potentially of bat origin, has been reported to cause respiratory tract infections among human beings in Southeast Asia. Twelve IgG ELISA-positive cases with antibodies against Pteropine orthoreovirus were detected among 272 human serum samples collected between March and June 2014 from in and around Hue City, Central Vietnam. These 12 cases were IgM ELISA negative. Neutralizing antibodies were also detected among six of these cases with the highest titer of 1:1,280 in 2 cases (both female, 32 and 68 years old, respectively). This is the first report of human infection with Pteropine orthoreovirus in Central Vietnam. These findings indicate the need for surveillance on Pteropine orthoreovirus infections in Southeast Asia to enable prevention and control strategies to be developed should a change in virulence occur.

A novel screening method for influenza patients using a newly developed non-contact screening system.

OBJECTIVES: In places of mass gathering, rapid infection screening prior to definite diagnosis is vital during the epidemic season of a novel influenza. In order to assess the possibility of clinical application of a newly developed non-contact infection screening system, we conducted screening for influenza patients. MATERIALS AND METHODS: The system is operated by a screening program via a linear discriminant analysis using non-contact derived variables, i.e., palmar pulse derived from a laser Doppler blood-flow meter, respiration rate determined by a 10-GHz microwave radar, and average facial temperature measured by thermography. The system was tested on 57 seasonal influenza (2008-2009) patients (35.7 degrees C < or = body temperature < or = 38.3 degrees C, 19-40 years) and 35 normal control subjects (35.5 degrees C < or = body temperature < or = 36.9 degrees C, 21-35 years) at the Japan Self-defense Forces Central Hospital. RESULTS: A significant linear discriminant function (p < 0.001) was determined to distinguish the influenza group from the control group (Mahalanobis D-square = 6.5, classification error rate > 10%). The system had a positive predictive value (PPV) of 93%, which is higher than the PPV value (PPV < or = 65.4%) reported in the recent summary of studies using only thermography performed mainly in hospitals. CONCLUSIONS: The proposed system appears promising for application in accurate screening for influenza patients at places of mass gathering.