How do you know whether disease X is the result of infection by pathogen
Y?
In 1890 Robert Koch published a list of 'postulates' which would form
the foundation of ascribing disease causation from that day - until now. Though
Koch established his list based upon his studies of bacteria, more
specifically Mycobacterium tuberculosis and Bacillus anthracis, (the causative agents
of tuberculosis and anthrax respectively), his postulates could be applied
more widely. No more would infectious diseases be merely regarded as mysterious
happenings. Scientists could now pursue and nail down the cause of a disease by
fulfilling Koch's postulates. There are many ways of expressing the same thing,
but Wikipaedia describes them as:
1) The microorganism
must be found in abundance in all organisms suffering from the disease, but
should not be found in healthy organisms.
2) The microorganism
must be isolated from a diseased organism and grown in pure culture.
3) The cultured
microorganism should cause disease when introduced into a healthy organism.
4) The microorganism
must be reisolated from the inoculated, diseased experimental host and
identified as being identical to the original specific causative agent.
Association vs. causation will always be a key issue when it comes to
diagnosis and Koch's postulates, as important as they are, soon start to fall
apart under closer scrutiny. For instance, what happens in circumstances where
the organism can't be isolated in culture - a not unusual occurrence in the
world of virology?
The most significant problems occur though simply due to words such as
'must'. Infectious disease is never so clear cut. Koch himself acknowledged as
much during his studies of cholera; whilst the causative agent, Vibrio cholerae, could be isolated
from people with cholera, it could also be isolated from healthy people. This
fails the first postulate (depending on how loosely you apply 'should').
What about the same agent in different species? The dogma with
Bluetongue virus is that it is asymptomatic in cattle, but can be lethal in
sheep. Even with sheep though, only a few of those which become infected
actually show bluetongue disease. The fact that the term 'case fatality' exists is an
acknowledgement itself that only a fraction of animals which become infected fall
ill. You could argue that this is an irrelevance as, even if it doesn't cause
disease in every animal that it infects, it is
still the causative agent of those which do become sick. Even though there
haven't been enormous numbers of cases reported (presumably because there's no
evidence of disease), I would put a lot of money on the fact that a large
proportion of cows in southern England are currently seropositive for Schmallenberg
virus.
In turn, when attempts are made to fulfill postulate 3, it
would be perfectly normal, expected even, that an animal doesn't become sick.
Discussions of these limitations are not new. The biggest development
that has prompted the questioning of Koch's postulates has been molecular
biology. The extreme sensitivity of nucleic acid-based methods means that
evidence of pathogens can be detected at extremely low levels. Now it is possible to go searching for viruses with relative ease,
increasing the speed and efficiency of virus detection and discovery. There are
many approaches; PCR is arguably the most common, and metagenomics the most
recent. I compare the methods to fishing: firstly, it’s possible to go fishing
for specific viruses using PCR. Metagenomics on the other hand is more like a
deep sea trawler – sequencing everything within a sample and looking within the
‘catch’ for virus sequences. Using the latter approach has allowed the
identification of numerous viruses, and this is reflected in the number of
publications, as revealed in a paper by Mokili et al. (2012).
The paper by Mokili et al. (2012) also
discusses the fact that molecular approaches remove the need for the growth of
an agent in pure culture. Significantly, there is also the acknowledgement that
determining causality merely by a pathogen’s presence is difficult to achieve,
particularly with metagenomics approaches which are capable of finding a
diverse array of agents within the same sample. The authors go on to describe a
‘metagenomic Koch’s postulates' approach whereby the metagenomes of individuals are
compared. The postulates are:
1) The diseased metagenome must be significantly different from the healthy
control and contain a greater abundance of the suspected metagenomic traits.
2) Inoculation of a
healthy individual with a sample from the diseased individual must result in
disease state.
3) Selected, specific samples containing the suspected traits from the
individual infected for step 2 must cause disease when injected into another
healthy individual.
Following this procedure allows the sequence associated with disease,
i.e. a biomarker of the etiological agent, to be discovered by the process of
elimination.
Metagenomics will, rightly, more than likely become established as the method of choice for diagnosis. The technology is still developing, but not too far in the future I suspect metagenomics will follow a similar path to that of real-time PCR into the molecular diagnostics setting. Instead of testing a samples against some 'likely suspects' using PCR approaches, it will be possible to get a complete picture of the complex 'virome' associated with that sample.
Metagenomics will, rightly, more than likely become established as the method of choice for diagnosis. The technology is still developing, but not too far in the future I suspect metagenomics will follow a similar path to that of real-time PCR into the molecular diagnostics setting. Instead of testing a samples against some 'likely suspects' using PCR approaches, it will be possible to get a complete picture of the complex 'virome' associated with that sample.
Although the 'metagenomics Koch's postulates' are a step towards linking metagenomics to disease, there is still the issue of ‘must cause disease’; if this ‘disease’ by definition involves clinical signs, then similarly to Koch’s original
postulates this may fail. However, the approach does allow the picking apart of
causality in complex scenarios where multiple pathogens are present. There are
still issues to be ironed out as to how molecular data is interpreted, but this is going to become increasingly important
as metagenomic approaches become even more widespread. From this perspective,
it appears that Koch’s postulates, landmarks and revolutionary as they may have
been, may be nearing the end of their life.
Mokili, J., Rohwer, F., & Dutilh, B. (2012). Metagenomics and future perspectives in virus discovery Current Opinion in Virology, 2 (1), 63-77 DOI: 10.1016/j.coviro.2011.12.004
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