Obtaining rapid and reliable diagnosis of infectious diseases is usually limited by the sensitivity of the detection technology. Even in severe sepsis, accompanied by organ failure and admission to an intensive care unit, the causative organism is often present at a level of less than one bacterium per milliliter of blood. Similarly, in candidiasis the yeast cells are present at vanishingly low levels in body fluids, while in chlamydia infections the pathogen is located intracellularly as is entirely absent from the blood fluid.
All these (and many other) pathogens have evolved to escape detection by the immune system, and its antibody sensors. This, coupled with the low levels of organisms in samples from infected individuals, means that antibody-based diagnostic tests rarely have enough sensitivity to be useful.
Then came PCR. The big selling point of the polymerase chain reaction is its exquisite sensitivity, while retaining useful specificity. Under optimal conditions you can detect a single DNA molecule with this technique. Surely PCR was going to revolutionize infectious disease diagnosis?
Not really. There are several problems: the very low levels of infectious organisms in the samples means that there is a very large amount of other DNA (from the host cells) in the sample. Unless some kind of enrichment is performed, the PCR reaction cannot achieve the necessary sensitivity in the presence of so much competing DNA template. Secondly, DNA from dead organisms is detected just as efficiently as from live ones, and worse still DNA released from the dead organisms can persist in the blood for weeks and months. Together, these issues lead to high rates of both false positive and false negative findings, and for many infectious diseases such simple PCR tests perform too poorly in the clinic to be of value.
A common solution that deals with both these problems is to culture the sample prior to running the test. The rapid growth of the infectious organism enriches the sample with the target DNA template, and at the same time differentiates viable organisms from dead ones. PCR on cultured samples usually achieves the necessary sensitivity and specificity to be clinically useful – but for severe disease, such as sepsis, the time taken to culture the sample (which may be several days) is critical when the correct treatment needs to be started immediately.
As a result, there is still a massive product opportunity for new infectious disease diagnostics.
One approach is to try and confer on the PCR tests the specificity for live organisms, and at the same time improve the ability to distinguish template from the organism from the high levels of host DNA. A particularly promising solution from Momentum Biosciences is to employ the DNA ligase enzyme from live bacteria to ligate added DNA template to create an artificial gene that is then amplified by conventional PCR. The product is still in development, but it offers real hope of a sepsis test that can identify live organisms in less than 2 hours.
But another potential solution comes from a much more surprising approach: using nuclear magnetic resonance (NMR) spectroscopy. NMR offers exquisite specificity to distinguish molecules in a sample based on their chemical structure, a property that underpins the use of the technique in metabolic profiling. However, as anyone who has ever tried to exploit this elegant specificity will tell you, the problem with NMR is its lack of sensitivity. Even with cutting-edge equipment, costing millions, the sensitivity limit is usually above 10µM (which equates to a million million or so molecule per milliliter of sample. Not much use, one might think, for detecting a single cell in a milliliter of blood.
But T2 Biosystems, based in Lexington, MA, have found a neat solution to the sensitivity problem of both antibodies and NMR. By coating highly paramagnetic beads with antibodies specific for the infectious organism, they can readily detect the clumping of these beads in the presence of very low levels of antigen. Again, the test is in development, but the company announced last week the closing of a $23M series D investment to bring the system to market.
There is an attractive irony in using a technique famed for its ultra-low sensitivity to solve a problem where sensitivity of detection was the limiting factor. In the race to find clinically useful diagnostic tests for many infectious diseases, just as in Zeno’s race between the hare and the tortoise, the super-sensitive PCR took a massive early lead and for a long time looked like the only winner in an arena where the major barrier to success was sensitivity of detection. But the wily old tortoise is not out of it yet: an ingenious twist added to low-sensitivity NMR might still win the race to clinical and commercial success in the infectious disease diagnostic arena.
Dr. David Grainger
CBO, Total Scientific Ltd.
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