Abstract
Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.
Highlights
Pathogenesis caused by intracellular pathogens, such as Mycobacterium tuberculosis among others, relies on the survival of the microorganism within host cells, such as macrophages and dendritic cells
DNA from peripheral leukocytes found in the buffy coat (Direct nested PCR (nPCR)) or leukocyte-derived cultured bacteria (Culture-based nPCR) is first isolated in a multistep protocol [21], yielding bacterial DNA of high quality (Figure 1a)
Based on the observation that the sample’s T2 times were higher than those of the sterile control throughout the experimental time course, we deduced that the hybridizing magnetic nanosensors (hMRS) first bound to the relaxed circular Mycobacterium avium spp. paratuberculosis (MAP) DNA that was heated, while the hMRS still remained bound to the cooled DNA as it was returning to its supercoil form
Summary
Pathogenesis caused by intracellular pathogens, such as Mycobacterium tuberculosis among others, relies on the survival of the microorganism within host cells, such as macrophages and dendritic cells. Serological immunoassays cannot effectively detect intracellular pathogens in biological fluids (e.g. blood, lymphatic fluid) as the microorganism is hidden away within the immune cells For this reason, isolation of the infected cells and extraction of their DNA, followed by detection of specific bacterial genomic markers via Polymerase Chain Reaction (PCR), is needed to facilitate identification of the pathogen. Bacterial culturing methods require a significant amount of time, such as in the case of slowgrowing intracellular pathogens that have to be cultivated for even weeks, assuming that there is an adequate amount of viable pathogens in the clinical sample Another drawback of culturing methods is that some pathogens do not grow effectively in culture, either because of the nature of the pathogen itself or the presence of biological interferences present in the clinical sample that prevent growth in culture. New technologies that can detect the presence of a pathogen in human clinical samples are urgently needed
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