Development of a Syndromic Molecular Diagnostic Assay for Tick-Borne Pathogens Using Barcoded Magnetic Bead Technology.

Infectious disease diagnostics often depend on costly serological testing with poor sensitivity, low specificity, and long turnaround time. Here, we demonstrate proof of the principle for simultaneous detection of two tick-borne pathogens from a single test sample using barcoded magnetic bead technology on the BioCode 2500 system. Specific primer sets complementary to the conserved genes of Anaplasma phagocytophilum and Borrelia burgdorferi were used in PCR amplification of the target, followed by the hybridization of the resulting biotinylated PCR products with specific probes tethered to the barcoded magnetic beads for simultaneous detection, using a fluorophore with high quantum yield. The assay has an extremely high signal to background ratio, with a limit of detection (LOD) of 2.81 50% tissue culture infection dose (TCID50)/mL and 1 CFU/mL for A. phagocytophilum and B. burgdorferi, respectively. The observed LOD for gene blocks was 1.8 copies/reaction for both the pathogens. The assay demonstrated 100% positive and negative agreement on performance evaluation using patient specimens and blood samples spiked with 1 × LOD of pathogen stock. No cross-reactivity was observed with other related tick-borne pathogens and genomic DNA of human, cattle, and canine origin. The assay can be upgraded to a sensitive and cost-effective multiplex diagnostic approach that can simultaneously detect multiple clinically important tick-borne pathogens in a single sample with a short turnaround time. IMPORTANCE The low pathogen load in the tick-borne disease test samples and the lack of highly sensitive multiplex diagnostic approaches have impacted diagnosis during clinical testing and limited surveillance studies to gauge prior insight about the prevalence of tick-borne infections in a geographical area. This article demonstrates proof of the principle for simultaneous detection of two important tick-borne pathogens from a single test sample using digital barcoded magnetic bead technology. Using a fluorophore of high quantum yield, the diagnostic approach showed high sensitivity and specificity. The LOD was 1.8 genome copies per reaction for both A. phagocytophilum and B. burgdorferi. The assay can be upgraded for the detection of all clinically important tick-borne pathogens from a single patient sample with high sensitivity and specificity. The assay can provide a diagnostic answer to the clinician in a short turnaround time to facilitate speedy therapeutic intervention to infected patients and implement public health measures to prevent community spread.

Current and Future Molecular Diagnostics of Tick-Borne Diseases in Cattle.

Ticks and tick-borne diseases such as babesiosis, anaplasmosis, ehrlichiosis, Lyme disease, Crimean Congo hemorrhagic fever, and Rocky Mountain spotted fever pose a significant threat to animal and human health. Tick-borne diseases cause billions of dollars of losses to livestock farmers annually. These losses are partially attributed to the lack of sensitive, robust, cost effective and efficient diagnostic approaches that could detect the infectious pathogen at the early stages of illness. The modern nucleic acid-based multiplex diagnostic approaches have been developed in human medicine but are still absent in veterinary medicine. These powerful assays can screen 384 patient samples at one time, simultaneously detect numerous infectious pathogens in each test sample and provide the diagnostic answer in a few hours. Development, commercialization, and wide use of such high throughput multiplex molecular assays in the cattle tick-borne disease surveillance will help in early detection and control of infectious pathogens in the animal reservoir before community spread and spillover to humans. Such approaches in veterinary medicine will save animal life, prevent billions of dollars of economic loss to cattle herders and reduce unwanted stress to both human and animal health care systems. This literature review provides recent updates on molecular diagnostics of tick-borne pathogens and discusses the importance of modern nucleic acid high throughput multiplex diagnostic approaches in the prevention of tick-borne infection to livestock.

Ultrasound evaluation of stress injuries and physiological adaptations in the fingers of adolescent competitive rock climbers.

BACKGROUND: The impact of high-intensity, repetitive training on the fingers of adolescent climbers is relatively unknown. OBJECTIVE: To evaluate this effect by ultrasound (US) and to confirm some findings by magnetic resonance imaging (MRI). MATERIALS AND METHODS: The US study was performed in 20 adolescent rock climbers (ages 10-17 years) and 6 non-climbing controls (ages 11-15 years). US was used to examine the third digit of the right hand for differences in thickness of soft tissue, flexor and extensor tendon, volar plate and bony and growth plate adaptations. In four climbers with finger deformity or pain, 3-T MR images were compared with US findings. Number of hours/week and years of climbing were used to group climbers in three levels (3=most intense training). Mann-Whitney test was used for statistical analysis. RESULTS: Compared with non-climbing controls, climbers demonstrated significantly thicker flexor tendons, volar plates and soft tissues. Joint effusions were found in 13/19 (68%) climbers. Significant phalangeal malalignment was seen in 10/19 (53%) climbers. Growth plate deformities were identified in three level 3 climbers. US findings correlated with MRI for effusions, phalangeal growth plate injury, malalignment and adaptive changes. MRI additional showed capsule rupture (n=1), stress fracture (n=1) and phalangeal physeal stress injury (n=1). CONCLUSION: Competitive rock climbing results in physiological adaptations in the fingers, an example being significant soft-tissue hypertrophy of the flexor. US demonstrated several non-physiological changes in response to repetitive stress in half of the climbers. MRI showed additional stress injuries to the growth plate, joints and bone.

The relationship between pre-verbal event representations and semantic structures: The case of goal and source paths.

We explored the nature of infants' concepts for goal path and source path in motion events (e.g., the duck moved into the bowl/out of the bowl), specifically asking how infants' representations could support the acquisition of the semantic roles of goal path and source path in language. The results showed that 14.5-month-old infants categorized goal paths across different motion events (moving to X, moving on Y), and they also categorized source paths if the source reference objects were highly salient (relatively large in size and colorful). Infants at 10months also categorized goal paths, suggesting that the broad concept GOAL PATH precedes the acquisition of the relevant spatial terms (e.g., "to", "onto"). These results are discussed in terms of the nature of goal and source path representations in infancy (e.g., whether they are represented at a general level - one that encompasses specific relations such as containment and support) as well as the possible mechanisms that may be involved in the mapping of these representations to language.