A very sensitive redox measurement
We are often asked: how is it that our technology can detect bacteria at such low numbers ?
Bacteria produce free radicals as part of their metabolic cycle (which alters the redox state of the test sample), and at it’s core, our technology is a very sensitive measurement of the redox (reduction-oxidation) state of test sample. We have developed methods that respond to very small amounts of free radicals. This approach is unique and will remain unique (because we have developed/patented the only possible amplification scheme that enables this sensitive/specific detection. It also has several advantages over other approaches. First, the effective free radical concentration can build up over time; and the concentration of free radicals can exceed bacteria concentration. Therefore, our test can detect the presence of pathogens in very small blood samples. All of our pilot studies have been done on either 0.3 or 0.6 mL of blood plasma; and we have results that are substantially in concordance with blood culture tests based on 10 mL blood draws. Second, we believe that our test is not spoofed by contaminants. About 1 in 3 positive blood cultures positives are positive due to a skin contaminant. These false positives result in significant diagnostic uncertainty, and is a significant problem for clinicians. In our test, skin contaminants have a minimal effect because they are not given the time required to produce free radicals. We do not yet have conclusive evidence of this, but the pilot study data is consistent with this expectation. Third, our test does not suffer from false negatives due to antimicrobial use. Antimicrobials that suppress bacterial cell growth can create false negatives in a blood culture test. We do not require growth, so we are substantially unaffected by antimicrobial use.
A very specific redox measurement
We are also often asked: Why is it that your test does not respond to other inflammatory processes?
All inflammatory processes produce free radicals. For instance, viral infections also result in free radicals being released into the bloodstream. Since our test responds to free radicals, it should generate a significant number of false positives, corresponding to all of those inflammatory processes.
But our test does not respond to those free radicals ~ it only responds to free radicals produced by bacterial cells. Bacterial cells have a known interaction that enables the preservation of free radicals produced by bacteria. By contrast, free radicals produced by other inflammatory processes are “scavenged” by various scavengers (primarily ascorbic acid and glutathione) present in the solution. It is possible that for very sick patients (i.e, those that are close to death), the scavengers are depleted, and in that scenario, we do anticipate a false positive on our test.
Antimicrobial susceptibility measurements
How do we characterize antimicrobial susceptibility ?
We characterize the functional antimicrobial susceptibility of the causative pathogen without going through the time consuming steps of culture-isolation, and without the time consuming step of waiting for growth. This is a big deal because the time consuming steps required for culture-isolation delays therapy optimization, and degrades patient outcomes. Further, we are characterizing the functional antimicrobial susceptibility, and not looking for a marker of resistance ~ this is a big deal because not all resistance mechanisms are known/catalogued, not all known resistance mechanisms have a marker, and because new mechanisms are continually developing. For these reasons, the functional antimicrobial susceptibility continues to be the gold standard. How do we do all that ?
Our test does responds free radical, and free radical production is modulated by the presence of an effective antimicrobial.
How do we identify the bacteria ?
The mechanisms described above are generic to any microorganism. But subtle variations on the above enables the detection of specific enzymes produced by certain bacteria, while preserving the inherent amplification schemes that enable the limit of detection. With a large enough library of such enzymes, we can “phenotype” the bacteria down to the species level. We are currently working on expanding the library of enzymes recognized by our test.
Incorporating insoluble molecules into albumin
We have developed methods by which any hydrophobic molecule can be incorporated into human serum albumin without invoking any bonds between albumin and the incorporated molecule. This is a big deal ! Several other groups tried various approaches for this, but they all ended up with a chemical bond between the incorporated molecule and albumin and/or bonds between the albumin itself, resulting in albumin nanoparticles. This is a big deal because our formulation also enables an ideal drug delivery platform. We are currently working on patent applications that would give us some protection on this drug delivery platform
Our reagent includes several photosensitive components. As such, they would normally be expected to degrade quickly. Indeed, if we manufacture our reagent using feedstock purchased from suppliers like Aldrich, then our reagent has a lifetime of about 6 hours. We have developed methods by which our reagent has a lifetime of >1 year.
In addition, our reagent is designed to respond to a single bacterial cell, so it is very sensitive to contamination. We have developed methods by which our reagent can be packaged and shipped using common carriers (DHL, FedEx etc) without being contaminated.
Separating Raman and fluorescence peaks
We measure the Raman spectrum using 532 nm light. At these wavelengths, the incoherent scattering output is dominated by the fluorescence contribution. We have developed methods to mathematically separate the Raman and fluorescence contributions. Our methods start with the Lieber method, but are refined to reduced mathematical artifacts.
The mathematical algorithms used to separate Raman and fluorescence are very sensitive to various interference effects from within the Raman spectrometer. These interference effects can vary from instrument to instrument, and can also vary within one instrument over time. We have developed methods to calibrate these factors. Thus, the result from one instrument can be mapped to another instrument.
Antimicrobial drug delivery
Given the powerful technologies developed above, there should be a number of other applications that are possible. Right ?
Indeed, we are able to incorporate nominally insoluble drug molecules into albumin, and then exploit the inherent properties of the system to deliver the drug directly to the surface of pathogenic microorganisms. This effect concentrates the drug onto the surface of the bacteria, which has the net result of decreasing the amount of drug required to kill the bacteria. In technical terms, we observe a reduction in the “minimum inhibitory concentration” MIC of that particular antimicrobial drug. We have verified this effect on a number of system; examples include Amphotericin B (against C. auris and C. albicans), and Clofazamine (against Gram Positive organisms); wherein we observe a 3x reduction in MIC. For Clofazamine, we also have initial data from an animal model verifying a 3x increase in killing efficiency.