Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

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

Clinical data and hMRS results of minimally processed blood samples (Cohort 2).

<p>Clinical data and hMRS results of minimally processed blood samples (Cohort 2).</p

Specificity and sensitivity of the IS900-detecting hMRS.

<p>(<b>a</b>) hMRS specifically bind to MAP DNA and not DNA from other mycobacteria or common pathogens ([hMRS]  =  3 µg Fe/µL). hMRS facilitates the fast and quantitative detection of MAP DNA in pure (<b>b</b>) and crude (<b>c</b>) samples within 30 minutes. Comparison of the sensitivity of the hMRS and nested PCR (nPCR). (<b>d</b>) Within 30 minutes, the hMRS detected a single MAP genome copy, translating to one bacterium. (<b>e</b>) After its second amplification round (6 hours), nPCR achieved comparable sensitivity. (Upper gel image: first nPCR round (3 hours), lower gel image: second nPCR round (total two-round time 6 hours), - Ctrl: dH₂O and 0: sterile TE buffer).</p

Screening of clinical isolates with hMRS and nPCR.

<p>Comparison between the magnetic relaxation-mediated detection of MAP in clinical isolates using hMRS after crude DNA extraction and culture-based nPCR. All isolates were from blood samples from Crohn’s disease patients, apart from GN2’ and GN8’ that correspond to ileal biopsies.</p

Detection of <i>Mycobacterium avium</i> spp <i>paratuberculosis</i>’ genomic marker in clinical samples with hMRS and direct nPCR.

<p>The hMRS detected MAP’s IS900 region in minimally processed blood samples (Cohort 1, n  =  34). Correlation of the hMRS findings was achieved through pure DNA extraction from white blood cells and direct nPCR. Results are means ± SE.</p

Design of a nanoparticle-based assay for the identification of genomic markers of the intracellular pathogen <i>Mycobacterium avium</i> spp. <i>paratuberculosis</i> (MAP).

<p>(<b>a</b>) Isolation of MAP requires collection of infected white bloods cells from blood samples via centrifugation. For direct nPCR analysis, DNA directly isolated from white blood cells is purified in multiple steps prior to amplification and detection by gel electrophoresis. Meanwhile, culture-based nPCR requires the growth of MAP in specialized liquid media for 12 weeks, followed by DNA isolation before nPCR. Hybridizing magnetic relaxation sensors (hMRS) can detect MAP DNA in minimally processed blood samples via changes in magnetic signal (ΔΤ2) in 1 hour, as opposed to 24 hours for direct nPCR and 12 weeks for culture nPCR. (<b>b</b>) Preparation of MAP DNA for hMRS. Heating of infected leucocytes facilities rupture of the cell membrane releasing MAP DNA. Further heating and cooling steps facilitates the annealing and binding of a MAP specific hMRS, resulting in an increase in the T₂ water relaxation time.</p

Detection of <i>Mycobacterium avium</i> spp <i>paratuberculosis</i>’ genomic marker in clinical samples with hMRS and culture-based PCR.

<p>(<b>a</b>) Blood samples from a second population (Cohort 2, n  =  26) were screened for the presence of the MAP IS900 genomic element with hMRS, direct nPCR and culture-based nPCR after 12-week cultivation. The receiver operating characteristic (ROC) curve indicated that the hMRS, can better differentiate between symptomatic (Chrohn’s) and asymptomatic (healthy) states than the two gold standard methods. (<b>b</b>) Two-proportion z-test confirmed hMRS as a better clinical diagnostic utility than the PCR-based methods.</p

Comparison between the hMRS genomic detection of MAP in clinical isolates and homogenized tissues with the presence of MAP protein markers using anti-MAP antibodies conjugated MRS.

<p>(<b>a</b>) hMRS detected MAP genomic tags in clinical isolates via changes in the magnetic resonance signal. (<b>b</b>) A second preparation of magnetic nanosensors carrying polyclonal anti-MAP antibodies (anti-MAP-pAb) was able to corroborate the presence of MAP in these clinical isolates by the identification of MAP surface epitopes. (<b>c</b>) hMRS detected MAP’s IS900 in homogenized tissue from Johne’s disease cattle. (<b>d</b>) Corroboration of the presence of MAP in the cattle samples using anti-MAP-pAb MRS nanosensors.</p