Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

BACKGROUND: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for "naked eye" end-point detection when testing 'real-world' clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. RESULTS: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91-100%. CONCLUSIONS: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing 'real-world' samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

Feasibility study of a point-of-care positron emission tomography system with interactive imaging capability.

PURPOSE: We investigated the feasibility of a novel positron emission tomography (PET) system that provides near real-time feedback to an operator who can interactively scan a patient to optimize image quality. The system should be compact and mobile to support point-of-care (POC) molecular imaging applications. In this study, we present the key technologies required and discuss the potential benefits of such new capability. METHODS: The core of this novel PET technology includes trackable PET detectors and a fully three-dimensional, fast image reconstruction engine implemented on multiple graphics processing units (GPUs) to support dynamically changing geometry by calculating the system matrix on-the-fly using a tube-of-response approach. With near real-time image reconstruction capability, a POC-PET system may comprise a maneuverable front PET detector and a second detector panel which can be stationary or moved synchronously with the front detector such that both panels face the region-of-interest (ROI) with the detector trajectory contoured around a patient's body. We built a proof-of-concept prototype using two planar detectors each consisting of a photomultiplier tube (PMT) optically coupled to an array of 48 × 48 lutetium-yttrium oxyorthosilicate (LYSO) crystals (1.0 × 1.0 × 10.0 mm3 each). Only 38 × 38 crystals in each arrays can be clearly re-solved and used for coincidence detection. One detector was mounted to a robotic arm which can position it at arbitrary locations, and the other detector was mounted on a rotational stage. A cylindrical phantom (102 mm in diameter, 150 mm long) with nine spherical lesions (8:1 tumor-to-background activity concentration ratio) was imaged from 27 sampling angles. List-mode events were reconstructed to form images without or with time-of-flight (TOF) information. We conducted two Monte Carlo simulations using two POC-PET systems. The first one uses the same phantom and detector setup as our experiment, with the detector coincidence re-solving time (CRT) ranging from 100 to 700 ps full-width-at-half-maximum (FWHM). The second study simulates a body-size phantom (316 × 228 × 160 mm3 ) imaged by a larger POC-PET system that has 4 × 6 modules (32 × 32 LYSO crystals/module, four in axial and six in transaxial directions) in the front panel and 3 × 8 modules (16 × 16 LYSO crystals/module, three in axial and eight in transaxial directions) in the back panel. We also evaluated an interactive scanning strategy by progressively increasing the number of data sets used for image reconstruction. The updated images were analyzed based on the number of data sets and the detector CRT. RESULTS: The proof-of-concept prototype re-solves most of the spherical lesions despite a limited number of coincidence events and incomplete sampling. TOF information reduces artifacts in the reconstructed images. Systems with better timing resolution exhibit improved image quality and reduced artifacts. We observed a reconstruction speed of 0.96 × 106 events/s/iteration for 600 × 600 × 224 voxel rectilinear space using four GPUs. A POC-PET system with significantly higher sensitivity can interactively image a body-size object from four angles in less than 7 min. CONCLUSIONS: We have developed GPU-based fast image reconstruction capability to support a PET system with arbitrary and dynamically changing geometry. Using TOF PET detectors, we demonstrated the feasibility of a PET system that can provide timely visual feedback to an operator who can scan a patient interactively to support POC imaging applications.

Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR.

Traditionary multiplex asymmetric polymerase chain reaction (PCR) can be applied to detect multiplex target organisms simultaneously, but complex optimizations of primer concentrations and staggered additions of primers are required to achieve equal amplification of multiplex genes. To overcome this shortcoming, we propose a novel method based on multiplex asymmetric PCR and paper-based nucleic acid diagnostics (PBNAD). In the asymmetric PCR, a universal primer was introduced to break the bottlenecks of low sensitivity and self-inhibition among different sets of primers. Amplification using the novel multiplex asymmetric PCR boosted the quantity of single-stranded amplicons, and the amplified products contained the same sequence at the 5' end. Therefore, only one gold nanoparticle-based signal probe was needed for the simultaneous detection of three genes using the PBNAD platform, and the detection signals could be observed with the naked eye. With this highly efficient, novel multiplex asymmetric PCR, as little as 1 pg/µL genomic DNA can be detected. This method can also be applied to genotyping for reliable epidemiological investigations. This proof-of-concept study highlights the potential of the PBNAD platform for cost- and labor-effective applications in the detection of pathogenic bacteria.