Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Simple, quick and cost-efficient: A universal RT-PCR and sequencing strategy for genomic characterisation of foot-and-mouth disease viruses.

Foot-and-mouth disease (FMD) is a major contributor to poverty and food insecurity in Africa and Asia, and it is one of the biggest threats to agriculture in highly developed countries. As FMD is extremely contagious, strategies for its prevention, early detection, and the immediate characterisation of outbreak strains are of great importance. The generation of whole-genome sequences enables phylogenetic characterisation, the epidemiological tracing of virus transmission pathways and is supportive in disease control strategies. This study describes the development and validation of a rapid, universal and cost-efficient RT-PCR system to generate genome sequences of FMDV, reaching from the IRES to the end of the open reading frame. The method was evaluated using twelve different virus strains covering all seven serotypes of FMDV. Additionally, samples from experimentally infected animals were tested to mimic diagnostic field samples. All primer pairs showed a robust amplification with a high sensitivity for all serotypes. In summary, the described assay is suitable for the generation of FMDV sequences from all serotypes to allow immediate phylogenetic analysis, detailed genotyping and molecular epidemiology.

Quantifying the accuracy of automated structure segmentation in 4D CT images using a deformable image registration algorithm.

Four-dimensional (4D) radiotherapy is the explicit inclusion of the temporal changes in anatomy during the imaging, planning, and delivery of radiotherapy. One key component of 4D radiotherapy planning is the ability to automatically ("auto") create contours on all of the respiratory phase computed tomography (CT) datasets comprising a 4D CT scan, based on contours manually drawn on one CT image set from one phase. A tool that can be used to automatically propagate manually drawn contours to CT scans of other respiratory phases is deformable image registration. The purpose of the current study was to geometrically quantify the difference between automatically generated contours with manually drawn contours. Four-DCT data sets of 13 patients consisting of ten three-dimensional CT image sets acquired at different respiratory phases were used for this study. Tumor and normal tissue structures [gross tumor volume (GTV), esophagus, right lung, left lung, heart and cord] were manually drawn on each respiratory phase of each patient. Large deformable diffeomorphic image registration was performed to map each CT set from the peak-inhale respiration phase to the CT image sets corresponding with subsequent respiration phases. The calculated displacement vector fields were used to deform contours automatically drawn on the inhale phase to the other respiratory phase CT image sets. The code was interfaced to a treatment planning system to view the resulting images and to obtain the volumetric, displacement, and surface congruence information; 692 automatically generated structures were compared with 692 manually drawn structures. The auto- and manual methods showed similar trends, with a smaller difference observed between the GTVs than other structures. The auto-contoured structures agree with the manually drawn structures, especially in the case of the GTV, to within published interobserver variations. For the GTV, fractional volumes agree to within 0.2+/-0.1, center of mass displacements agree to within 0.5+/-1.5 mm, and agreement of surface congruence is 0.0+/-1.1 mm. The surface congruence between automatic and manual contours for the GTV, heart, left lung, right lung and esophagus was less than 5 mm in 99%, 94%, 94%, 91% and 89%, respectively. Careful assessment of the performance of automatic algorithms is needed in the presence of 4D CT artifacts.