NZ623074B2 - Biomarkers for respiratory infection - Google Patents
Biomarkers for respiratory infection Download PDFInfo
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- NZ623074B2 NZ623074B2 NZ623074A NZ62307412A NZ623074B2 NZ 623074 B2 NZ623074 B2 NZ 623074B2 NZ 623074 A NZ623074 A NZ 623074A NZ 62307412 A NZ62307412 A NZ 62307412A NZ 623074 B2 NZ623074 B2 NZ 623074B2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6421—Measuring at two or more wavelengths
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
Disclosed is a method of determining a level of activity of Pseudomonas aeruginosa bacteria in the lung of a patient, the method comprising: making a first measurement at a first time of a level of at least one marker of a bacterial iron scavenging process and of at least one secreted bacterial protein in a sample of sputum from the lung; making a second measurement at a second time of the levels of said marker and said protein in a sample of sputum from the lung; and determining said level of bacterial activity from changes in said measured levels of said marker of a bacterial iron scavenging process and said secreted bacterial protein over time, wherein an increase in levels of either said marker or said protein, or both, are indicative of an increase in bacterial activity. tein in a sample of sputum from the lung; making a second measurement at a second time of the levels of said marker and said protein in a sample of sputum from the lung; and determining said level of bacterial activity from changes in said measured levels of said marker of a bacterial iron scavenging process and said secreted bacterial protein over time, wherein an increase in levels of either said marker or said protein, or both, are indicative of an increase in bacterial activity.
Description
Biomarkers for respiratory infection
FIELD OF THE INVENTION
The present invention relates to a method for ining a level of activity of
bacteria in the lung of a patient. The method is intended particularly, but not
ively, for identifying the presence and level of activity of bacterial infection in
the lung of patients with cystic fibrosis; or for predicting the exacerbation of an
infection in such patients. Other s of the invention relate to s for
determining the effectiveness of a treatment of a bacterial lung ion.
BACKGROUND TO THE INVENTION
In the East of d region, 80.000-125,000 al bed days are required to
treat patients with atory infections each year. The most challenging patients to
treat are the most vulnerable: the elderly, neonates and those suffering from c
conditions such as cystic fibrosis (CF), Chronic Obstructive Pulmonary Disease
(COPD) or HIV. Respiratory infections in patients with chronic disease conditions
can be difficult to treat: infection with even the most common respiratory pathogens
may prove fatal.
Many patients with CF are colonised with one or more pathogens, the most
common being Pseudomonas aeruginosa. This gram-negative bacterium colonises
CF patients and evades all attempts at eradication. It undergoes numerous flare-ups
(exacerbations) and the inflammation it causes results in the permanent loss of lung
function. It also becomes resistant to antibiotics over time, making each subsequent
infection more difficult to control than the last. This is an adaptable, resilient and
lethal pathogen to be colonised with. The challenge for clinicians treating CF
patients is to reduce the number of infections and decrease the severity of each
ion. In doing so, lung on is preserved and life expectancy is increased.
For most CF patients, a lung infection with the resulting sepsis and le organ
failure, is the primary cause of death.
Infection with P. aeruginosa becomes problematic when there is an exacerbation of
infection, triggered by other factors. If not treated promptly and with the correct
antimicrobial medication, the t may be admitted to hospital for 2—4 weeks until
the infection can be controlled. This exacerbation and the lung mation which
follows can be anied by a dramatic and often permanent loss of lung
function and the rise in exotoxins produced by the pathogen, leading to sepsis.
At every stage in the treatment of ions, CF patients must travel to their clinic
and see their clinician as an outpatient for several consultations. Failure to control
infection as an outpatient results in a tay admission. Continued failure to
control the frequent infections (a CF patient may suffer 4—6 ions a year), will
cause irreparable damage to health which again, increases the cost of healthcare
over the life-time of the patient.
It is important to note that it is not merely the presence of an infection which is
adverse to ts. Many patients will have an ongoing, low level, infection which is
t to periodic exacerbations. Predicting the timing of these exacerbations is
significant for management of treatment. Simply ing the presence or absence
of bacteria - for example, by nucleic acid sequencing - will not in itself be informative
as to the likelihood of an exacerbation, as exacerbations can be triggered by many
factors.
it is known to use the presence of a biomarker, such as a secreted protein, as a
diagnostic for infection. For example, we have previously developed a simple
laboratory-based test to measure P. aeruginosa Exotoxin A (a well known marker of
infection by P. aeruginosa) in the patient's sputum.
But relying on a single biomarker for accurate assessment of the status of infection
is problematic. For e, products which detect other pathogens often only look
for toxins, such as the many rapid tests for C. difficile that are already on the
. These detect the presence of bacterial Toxins A and B in faecal samples.
While quick to perform (30 minutes), these rapid tests have low accuracy because
toxins A and B may not be produced during all infections or cannot be detected in a
3O given sample - this gives a low detection rate compared with the slower, but more
sensitive s of culturing cells from a sample by traditional microbiology (2-3
days). Furthermore, it is believed that bacterial populations may alter the profile of
toxins or other proteins ed over time in response to environmental factors;
within a given tion, there may be only some cells which produce a particular
protein while the remaining cells contribute to the infection but rely on these
exogenous ns for their survival. It is also known that a given population of
bacteria in a colonised patient mutate over time from the wild type with which the
host was originally infected. Accordingly, detecting a single n may not be
sufficiently accurate as a diagnostic of the likelihood of an bation.
To avoid this risk of poor accuracy, we have identified in the present invention
several biomarkers which can be measured quantitatively. in addition, we can also
profile biomarkers that indicate the status of the host's response to this pathogen.
We believe that taken together, a combination of these markers can be used to
detect an exacerbation before the patient feels unwell, thereby reducing the time to
ibe the first antibiotic and therefore reducing the severity of infection.
Martin et at, Biometals (2011) 24: 1059-1067 describes the detection of
siderophores produced by Pseudomonas aeruginosa in the sputum of patients with
cystic fibrosis. They found an association between presence of pyoverdine and
number of bacteria, but not in 21 out of 148 patients; and conclude that there is no
correlation between the amount of bacteria and clinical status. The authors also
conclude that the levels of siderophores do not markedly change during
exacerbations. This publication therefore teaches that profiling with siderophores
cannot be used to determine the level of virulence.
By contrast, as bed further below, the present inventors have determined that
siderophores are a usefui marker for bacterial exacerbations, when used in
combination with other s. We therefore provide an accurate and rapid test for
ining such exacerbations.
Jaffar-Bandjee et al., l of Clinical Microbiology, Apr 1995, p924-929 describes
the production of elastase, exotoxin A, and alkaline protease in sputa during
pulmonary exacerbation of CF in patients chronically infected by Pseudomonas
nosa. They found that the concentrations of exoproteins varied by patient on
admission (that is, after the exacerbation begins), but that the three proteins studied
(elastase, Exotxin A and alkaline protease) had similar levels. However, it is
apparent from data presented in the current application that different patients may
include different bacterial populations which produce different toxins or s.
Further, no test was made to detect tein levels prior to exacerbations.
ore profiling any of these exoproteins either alone or in combination will not
provide sufficient data to t all bations in all CF patients.
Further, with an objective and quantitative test, the treating clinician will be able to
quickly ine the performance of an antimicrobial medication in controlling the
infection, substituting one antibiotic for another if the first fails to bring the infection
under control. Typically, it can take up to 3 weeks to try different antibiotic
combinations in an iterative process, before an effective solution is established - this
is usually achieved by med guesswork" on the part of the expert clinician. With
our diagnostic test, we believe that the time taken to perform this most necessary
trial-and-error process could be reduced from 3 weeks to just 7 days.
For ts with CF, infection leads to inflammation of the lung and the r the
inflammation and time of inflammation, the greater the loss of lung function. Most
CF patients suffer 4 infections each year and can spend 50% of their time in
hospital. This could be reduced through the use of our new multi-marker test.
Many hospital admissions would be d if there was a rapid and accurate
ment of individuals with bacterial ions. One of the major difficulties
when ing patients with respiratory infections is to distinguish bacterial from
viral infections. Clinical features are frequently misleading and many patients
subsequently admitted to al had encountered delays in receiving antibiotics in
the community. In the USA approx 1-in-18 or 5.51 % or 15 million people per year
have misdiagnosed lower respiratory tract ions.
Moreover, primary healthcare workers also face the dilemma of selecting
appropriate antibiotics. While empirical antibiotic selection usually results in
satisfactory treatment, the y to identify the level of threat to a vulnerable patient
posed by an identified pathogen, would permit optimised antibiotic usage. This
would result in: improved early treatment success thereby preventing clinical
deterioration and subsequent hospital admission, and reduced use of broad
spectrum antibiotics.
In this specification, references to prior art are not intended to acknowledge or
t that such prior art is widely known or forms part of the common general
knowledge in the field either in New Zealand or elsewhere.
In this specification, the term ‘comprises' and its variants are not intended to exclude
the ce of other integers, components or steps.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention, there is provided a method of
determining a level of activity of Pseudomonas aeruginosa ia in the lung of a
patient, the method comprising:
making a first ement at a first time of a level of at least one marker of a
bacterial iron scavenging process and of at least one secreted bacterial n in a
sample of sputum from the lung;
making a second measurement at a second time of the levels of said marker and
said protein in a sample of sputum from the lung; and
determining said level of bacterial activity from changes in said measured levels of
said marker of a ial iron scavenging process and said secreted bacterial
protein over time, wherein an increase in levels of either said marker or said protein,
or both, are indicative of an increase in bacterial activity.
We have determined, surprisingly, that changes in levels of markers of bacterial iron
scavenging ses, such as siderophores, can be predictive of exacerbations,
but not in all patients. Similarly, changes in bacterial toxin levels can also be
predictive in some patients. A reliable test can be obtained by combining the two
measurements as described herein. Further, due to individual patient variability, it is
desirable to measure changes over time rather than absolute levels at a single time
point; some patients may live with higher background levels than others, and so it is
the changes which are diagnostic.
P. aeruginosa, like other pathogens, es Fe (III) ions to survive. The lungs of
CF patients typically produce large amounts of mucus, but also leak blood into the
fluid-coated air spaces of the lungs. This serves as an iron source for the pathogen.
P. 'nosa has multiple mechanisms whereby Fe (III) may be scavenged, and
of
appears to change from one ism to another depending on the condition
the host. However, we believe that it is possible to use levels of markers of iron
scavenging processes as markers of bacterial activity.
Preferably the marker of an iron ging process is a marker of an Fe (III)
scavenging process.
Preferably the marker of an iron ging process is a siderophore. ln preferred
embodiments, the siderophore may in ular include one or more of: pyochelin,
pyoverdin and pyocyanin.
The marker is a bacterial marker, although in n embodiments markers of host
iron scavenging processes may also be detected. In healthy patients, Fe (III) ions
are scavenged by the host (eg, by lactoferrin, ferritin, or transferrin) to prevent free
Fe (lll) lating which would serve as a reservoir for bacterial growth. Thus the
presence of host markers may be useful as an onal marker for determining
bacterial activity.
The secreted bacterial protein is preferably a toxin, and may be exotoxin A.
Alternatively, the secreted protein may be elastase or alkaline protease or indeed,
any protein secreted by the bacterium.
In a preferred embodiment, the combination of pyoverdin and in A are
detected.
The method preferably further comprises measuring at least one additional marker.
The additional s may be selected from bacterial toxins, host iron scavenging
markers, bacterial iron scavenging markers, and host inflammatory s. We
believe that detecting a combination of additional markers provides greater
sensitivity, accuracy and reliability to the assay than detecting the combination only
of markers of iron scavenging and ed toxins In a particularly preferred
embodiment, the combination of a bacterial toxin, a ial marker of iron
scavenging process, and a host marker of an iron scavenging s are detected.
For example, exotoxin A, a siderophore, and haem or a haem breakdown product
may be detected
The inflammatory marker may be a cytokine.
In certain embodiments, the marker has a concentration dependent on a level of
quorum ling between P. aeruginosa bacteria.
The patient may be a patient with cystic fibrosis. In other embodiments, the patient
may be a t with a chronic lung condition, for example, Chronic Obstructive
Pulmonary Disease (COPD).
The method may further comprise measuring the level of a plurality of markers of
iron scavenging processes and determining the level of bacterial activity from the
plurality of measured levels; the markers may be bacterial s, host markers, or
a combination of both.
The method may r se measuring the level of at least one iron (lll)
sequestration intermediate for determining said level of bacterial activity.
The method may further comprise the step of making additional measurements at
further time points
In a further aspect, the present invention provides a method of predicting an
exacerbation of a level of bacterial activity in the lung of a patient, the method
comprising making a time series of measurements of bacterial activity on said
patient using a method comprising making a measurement of a level of at least one
bacterial marker of an iron scavenging process and of at least one secreted
ial protein in a sample of sputum from the lung; and determining said level of
bacterial activity from said measured level of said marker of an iron scavenging
process and of said protein;
and using said time series of measurements to predict said exacerbation,
wherein an increase in measured activity over the time series is indicative of an
exacerbation.
A still further aspect of the present ion provides a method of determining the
effectiveness of a treatment of a bacterial lung ion, the method comprising
making a time series of measurements of ial activity on said t using a
method comprising making a measurement of a level of at least one bacterial
marker of an iron scavenging process and of at least one secreted bacterial protein
in a sample of sputum from the lung; and determining said level of bacterial ty
from said measured level of said marker of an iron scavenging process and of said
and determining said effectiveness from a time profile of said level of bacterial
activity, n a decrease in measured activity over time series is indicative of
effectiveness of the treatment.
Preferably the treatment is an antibiotic treatment. Preferably the method comprises
determining that said antibiotic ent is ineffective if said level of bacterial
activity does not fall with time.
A further aspect of the ion may e a device for use in any of the methods
of the invention, the device comprising:
means for collecting said sample of sputum;
means for making a measurement of levels of at least one bacterial marker of
an iron scavenging process and of at least one secreted bacterial protein in said
The device may further se means for ining said level of bacterial
activity from said measured levels of said at least one marker of said iron
scavenging process and said protein.
The means for making a measurement of levels may include a reagent which binds
to the relevant marker and a reagent which binds to the relevant protein. For
example, the reagents may se antibodies. The device may comprise a lateral
flow strip including said reagents; and may further comprise a colour change marker
to indicate the levels of said marker and/or protein.
BRIEF DESCRIPTION OF THE DRAWlNGS
Figure 1 shows an illustration of quorum sensing in P. nosa bacteria. Quorum
sensing between bacteria allows cells to communicate chemically. HHQ, the water-
soluble precursor of PQS is thought to be assembled in the periplasm (the space
n the cell membrane and the cell wall) from components synthesised in the
cytoplasm of the cell. HHQ from one cell is acted on by enzymes in the periplasm of
a second cell. It is believed that the active molecule, PQS forms vesicles of lipids
from the cell membrane of the second cell. When these vesicles ct with the
cell membrane of the first cell, it is t that this provides a signal to start the
production of the machinery required for cell growth and replication - in other words,
to increase the rate of metabolism. This initiates the production of toxins to kill-off
competing bacteria and the isms to scavenge all available free iron (Ill) ions
in the immediate vicinity through the use of siderophores and ferrioxidases. This
latter is an enzyme which converts iron (ll) into iron (III) that the cell needs.
Figure 2 shows a diagrammatic profile of the phases of infection in a CF patient and
their feeling of wellness. The patient usually only presents at the clinic when the
exacerbation has progressed into a full-blown infection.
Figure 3 shows the chemical structure for the siderophores identified from P.
aeruginosa - just one way in which this pathogen can e for iron (Ill). These
along with other Iron (Ill) sequestration intermediates such as Ferrioxidase, could be
used to fy exacerbations of ions in patients with CF.
Figure 4 shows the hypothetical profile of Exotoxin A and
Pyoverdin/Pyocyanin/ferrioxidase from P. aeruginosa.
Figure 5 shows the hypothetical profile of the levels of Exotoxin A and
Pyoverdin/Pyocyanin/ferrioxidase compared with patient wellness after
administration of the first antibiotic. It could be some days before the patient reports
to feeling well and this may not be a good reporter for the efficacy of the drug
stered in bringing the infection under control.
Figure 6 shows how the present invention may reduce the timelines for the empirical
evaluation of the antibiotic options available to the clinician. Our invention could
compress a 21 -day process into a 7-day s, simply by using a once-a-day
disposable test device.
Figure 7 shows a design t for a device for use with the method of the present
invention.
Figure 8 shows an emission spectrum from a typical patient for detection of
siderophores.
Figure 9 shows another emission spectrum illustrating the typical peak at 340 nm.
Figure 10 shows exotoxin A levels over time in patient 5.
Figure 11 shows 340 nm and 460 nm emission peaks over time in patient 2.
Figure 12 shows exotoxin, siderophore, and inflammatory marker levels over time in
patient 3.
Figure 13 shows exotoxin A levels over time in patient 3.
DETAILED PTION OF THE INVENTION
The natural habitat for the P. aeruginosa bacterium is not in human hosts - it lives in
water and survives in a planktonic state (free cells in water) in rivers and in soil. it
can thrive in this nment because it can rapidly adapt and is able to grow on
nearly any source of carbon-based foods. Even chemicals toxic to other bacteria
such as ol can be used by this tough pathogen as a carbon source to
support growth and replication (1 ). It adapts quickly under certain conditions, by
expressing a wide range of genes ated with metabolism and by rately
undergoing mutation at a very high rate.
Once established in our lungs (colonisation), P. aeruginosa adapts by changing the
way it lives. No longer in free suspension in the lungs, this ium secretes
mucus to form biofilms on the lung surface in which they then hide and protect
themselves from the host's own immune defences - and from antibiotics. This
makes it largely impossible to remove P. aeruginosa once a CF patient has been
colonised (usually in their mid , which means that this organism stays with the
CF patient for the rest of their life, becoming a parasite that ntly threatens the
life of the host as it undergoes rapid mutation and evolves into ever more virulent
forms.
Recent research in which bacterial DNA isolated ly from sputum was
sequenced (and therefore without introducing the bias of subculture), showed that
the 10-20 strains present in a single patient all shared the same ancestor and
derived from a single clone which then mutated. P. aeruginosa ore oes
adaptation through mutation, presumably in reSponse to the changes in the host as
the patient ages and their lung composition changes over time and as the lung
function decreases with each infection-induced inflammation. Each new strain which
takes hold probably has a particular advantage over its ancestor that makes it better
3O suited to this ever changing environment.
P. aeruginosa appears to live in a form of complex co-operation between cells of the
same strain and even between other strains. They have evolved a way of signalling
their metabolic status to other cells through a "chemical language”. Many chemical
signals have been ed and identified and are collectively called Pseudomonas
Quorum Signalling (PQS) molecules. These are complex molecular structures and
are often insoluble in water which means that they require complex laboratory
instrumentation such as LC MS to detect their presence in patient .
There are many ideas to explain the purpose of this cell-to-cell signalling, but the
most popular theory to explain why cells have developed this capability is that it
allows a cell to sense when others around it are increasing their metabolic activity
and are about to start a period of rapid multiplication - for the bacterium, this is
called Quorum Sensing (Figure 1 ). Perhaps the t to one strain of the
ium of being able to sense when the bacterial population is about to increase
rapidly, is that each strain can ensure that it is not mpeted for resources by
others or that its is not poisoned by the production of exotoxins from another
bacterium, by also accelerating its metabolism.
For the bacterium, Quorum Sensing precedes rapid multiplication. For the patient,
Quorum Sensing precedes an exacerbation. The trigger causing this change in the
bacterium's state from being subdued to highly active is unknown - perhaps through
a viral infection or some other change in the health of the t host. But once it
begins and if left untreated, an exacerbation will develop into a full—blown infection
within 2-4 days and often before the patient feels unwell (Figure 2).
The patient’s response to an exacerbation can also include an inflammatory
ion itself.
response, which can be as damaging to the patient‘s lungs as the
This provides another set of biomarkers which may be followed to objectively
assess the wellness state of the CF patient.
80 knowing when to treat a CF patient with otics so as to control an
exacerbation before it develops into a full ion, even before the patient feels
unwell, could reduce the severity of infection each time it happened. Also knowing
when to intervene through the application of antibiotics is a challenge for both the
clinicians and for the CF patient. Over-prescription of antibiotics leads to the
bacteria becoming resistant to antibiotics by evolving this ability sooner. For CF
patients, there are only 2 or 3 antibiotics useful in quelling a full—blown lung ion
- and so these medicines have to be prescribed only when essential. This means
they cannot be given as a lactic and taken continuously, as this would only
bring forward the time at which this pathogen develops the capability to tolerate
them.
There are few objective measures of wellness for a CF t as an exacerbation
event may be underway without the patient feeling ill. Classical methods used for
the non-quantitative monitoring of lung infections are: listening to breath sounds, X-
rays and ultrasound and the appearance of and culture of sputum (a mixture of
mucus, debris and cells expelled by the lungs). al staff experienced in
supporting CF patients have also become adapt at assessing the status of a patient
by observing the , viscosity and colour of a t‘s . The ability to
make such subjective observations is acquired from years of experience on the
ward - a highly specialised skill which also requires the patient to be at the clinic.
The unmet clinical need is for a rapid, portable, simple and low cost test which is
able to quantitatively measure "markers of exacerbation" in sputum, the
tration of which can be used to detect an exacerbation before the full
infection takes hold The detection of P. aeruginosa itself is of no benefit as it is
always present in most CF patients. For example, detecting the presence of P.
aeruginosa by qRT PCR is highly sensitive and quantitative for mRNA but this does
not correlate to the extent of sepsis, only the presence of the bacterium - and is too
expensive and complex to be used in a home environment anyway. What is needed
is the ability to detect the change in metabolic status of this ium before an
exacerbation converts into an infection, by following the tration of markers
longitudinally in a patient over time.
Aspects of the present invention provide a very simple multiple marker process
which has not yet been applied in this field: Combinations of any or all of the
following markers may be used; in certain embodiments, a single marker -
preferably a marker of an iron scavenging process - may be used as an initial
diagnostic. The markers are as follows:
Marker 1 - detect the toxins produced (eg Exotoxin A) as these are simple to test for
using a low cost immunoassay. Exotoxin A is produced by P. aeruginosa when it is
highly active and it secretes these complex les as natural poisons to ff
other bacteria and so gain an advantage in the competition for ces for its own
growth. Exotoxin A is also very toxic to the patient, causing symptoms of sepsis,
with multiple organ damage.
To increase the cy of our test, it would also be an advantage to detect other
markers in addition to Exotoxin A to quantify the "bacterial load" (an indication of the
total amount of bacteria present in the lungs and their activity “status"). The Quorum
Signalling les which communicate between ia would be ideal as this
would allow the clinician to "listen into the conversation between cells", but these
are water—insoluble and difficult to measure t the use of x laboratory
equipment. 80 we have to look for some thing else: a secondary marker that is
easier to measure but which is closely d to Quorum Signalling.
Marker 2. Iron scavenging markers from the pathogen. Despite its resilience, like all
living , P. aeruginosa must have iron (Ill) ions for it to thrive. This need gives
us the opportunity to find a new "handle" by which we can assess how active this
pathogen is at any given time, in addition to following the production of Exotoxin A.
This new handle or biomarker represents Marker 2 in our test - a handle on how
active the cell is in gathering iron (Ill) ions or more properly, kers for the
levels of activity of iron (Ill) sequestration by the bacteria within the lungs.
The lungs of CF Patients produce a lot of mucus but also leak blood into the fluid-
coated air spaces of this vital organ. With this plentiful supply of iron (lll), P.
aeruginosa is able to thrive in CF patients (2) and may even be the cause of
anaemia in CF patients (3).
P. aeruginosa has a multitude of different mechanisms by which it ters iron
(Ill) and it appears to change from one mode to another in response to the ever
changing condition of the host. It is therefore overly simplistic to select just a single
marker and so we will take a multiplexed approach.
Simple and easy markers for iron (Ill) sequestration activity assay: The t
markers to quantify are the bacterial enzyme co—factors or "siderophores" which are
produced in copious quantities prior to an exacerbation as a "secondary signal" -
that is, they are produced in high concentrations only during periods of active
growth. P. aeruginosa secretes a variety of pigments, including Pyocyanin (blue-
green), Pyoverdin (yellow-green and fluorescent), and Pyorubin (red-brown) (Figure
3). Under the microscope, P. aeruginosa is often preliminarily identified by its
pearlescent appearance and grape—like odour. Definitive clinical identification of P.
aeruginosa often includes fying the production of both Pyocyanin and
Pyoverdin, as well as its ability to grow at 42°.
These siderophores have a very high ty for the iron (Ill) ion and are thought to
be involved in the mechanism by which these bacteria absorb iron essential for
growth (4). The ability to secrete these iron scavengers into the immediate
environment around the cell gives the bacterium an advantage in that it can up
and trap" the iron it ately needs for rapid cell division and protein production.
Measuring the concentration of these coloured les could be an easy
biomarker of iron sequestration activity to profile.
Pyoverdin should be very easy to detect because it is fluorescent - this fluorescence
can be quenched by adding iron (III) ions. It can also be detected in sputum using a
competition reaction with an iron (IIl)-blnding dye called chrome azurol 8 (CAS)
reagent in a spectrophotometric assay (5). SiderOphores could also be detected
through the use of an immunoassay, although none have yet been developed.
Quantitation of Pyoverdin in sputum using optical ion (absorption or
fluorescence) requires extraction with solvents because the thick opaque mucus
interferes with the measurement and this does not lend itself to simple s for
home use, but can be used in our laboratory as we perform our initial feasibility
tests.
Indeed, Huston et a/ (6) have already undertaken similar studies in which they found
that the concentration of siderophores produced by cultures of bacteria isolated
from CF patients and grown in culture media, varied considerably between patients.
They profiled the concentration of the siderophores from isolates from a single
patient sample then cultured in the laboratory, rather than measuring the
concentration of siderophores directly in sputum and then ing a series of
samples from the same patient. Therefore they did not observe the relative s
in concentration for each patient, from a series of samples taken before, during and
after an exacerbation. Huston at al, working without the benefit of close co-operation
with an expert clinician, concluded that siderophores cannot be used as a ker
for exacerbation. This approach is flawed and is seen repeatedly in the scientific
literature. This arises because the biochemists undertaking the ch are
disconnected from their colleagues in the clinic. Studying the expression patterns of
cells grown in the test tube is spurious: we can make these cells do almost ng
we want them to by changing their growth conditions, and without the anchor of
clinical nce, this information is of little value in developing clinical tools. These
pathogens are adaptable - if the researcher changes their growth media, the cells
change their our. What is required is to go directly to the al sample and
solve the problem of how to measure the s in sputum rather than to use
cultures of cells produced in artificial media.
Huston et 8! ded that yet another iron (Ill) sequestration pathway ing the
bacterial enzyme ferrioxidase which is secreted into the periplasm of the bacterium,
was the correct biomarker to profile. What these biochemists missed was that it is
the total activity of the bacterium in gathering iron (III) that is the best biomarker and
this preferably requires a multiplexed approach if we are to create a tool le for
all patients, with their different strains, all of which are at different stages of
evolution within their hosts.
As well as the two preferred markers, toxins and iron ging markers, it may be
possible to include onal markers in the assay. These include:
Marker 3. Iron scavenging s by the host. In healthy people. our lungs prevent
iron (Ill) ions from accumulating in the fluid lining lung surfaces so that bacteria
cannot take-hold and grow. By absorbing iron (Ill) ions in number of ways including:
the production of iron-binding proteins called lactoferrin, ferritin and transferrin, we
protect ves by making it scarce to invading pathogens - a very effective form
of defence against disease. We also break down haem, the prosthetic group that
consists of an iron atom ned in the centre of a large heterocyclic organic ring
called a porphyrin. Haems are most commonly seen as components of hemoglobin,
the red pigment in blood, but they are also components of a number of other
hemoproteins. In competition with bacteria, the host produces the enzyme haem
mono oxygenase (aka Ferridoxinase) which breaks down haem into Iron ions (which
cannot be absorbed by the bacteria), biliverdin (a yellow t) and carbon
monoxide. By following the s in levels of either the enzyme or biliverdin in
sputum, we can determine the activity of the host's natural defences against this
pathogen.
Marker 4. Inflammatory response: cytokines. Cytokine markers have been reported
in the literature extensively. In particular, markers TNFa and lL-8 have already been
used to profile patents in our laboratories. Airway disease in cystic is characterised
by a uous cycle of chronic ion and inflammation dominated by a
neutrophilic infiltrate. This inflammation is characterised by an increased tion
of pro-inflammatory cytokines in the lung. The relationship between the abnormal
CFTR gene product and the development of inflammation and progression of lung
disease in CF is not fully understood. Courtney et a/ (7) review the mechanisms of
ary inflammation in CF, the profiles of cytokines and inflammatory mediators
in the lung in CF, and the mechanisms that may predispose to chronic P.
aeruginosa infection. Imbalances of cytokine secretion are now better understood
due to recent advances in understanding CF at a molecular level and it is
singly thought that the normal inflammatory process is deranged in CF early
in the course of the disease and may occur in the absence of detectable infection.
A combination of markers for greater accuracy.
By orating assays for bacterial markers for toxin production and iron
scavenging, in a simple, multiplexed test, we should be able to accurately assess
the metabolic status of P. aeruginosa in real-life sputum samples and hence, in the
lungs of the patient. Further, by measuring the response by the host in iron
scavenging AND cytokine-mediated inflammation, we have another objective means
for the determination of the CF patient‘s status. See table 1 .
Pathogen
Toxin production ;= Exotoxin A
Iron scavenging: Iron scavenging:
sidiarophores {b} Haem mono oxygenase
Haem mono city/genders [oi Biliverdin
Inflammatory response Inflammatory response:
Table 1 . Summary of multiple markers useful in the accurate longitudinal profiling of
CF patents
This approach resolves the low accuracy problem associated with rapid tests for C.
difficile that is described above. Indeed, this approach of understanding the
metabolic ys of a pathogen and identifying secondary reporters to be used in
ation with the ion of bacterial toxins, can be applied to many different
pathogens such as Staphylococcus aureus, a second pathogen common in CF
Our opinion is that the use of quantitative assays to profile the four classes of
ker, quantitatively and in longitudinal is of each patient, concentration
performed on untreated sputum (rather than an invasive blood sample or cells
cultured in presentative growth media) so as to ine the bacterial load
of P. aerug/nosa, is both accurate and sensitive. (Figure 4).
Our approach of quantifying these smaller kers is far easier to develop and
e into a device than the detection of the bacterium itself, which requires
proteins or other targets to be ed from cells or cell membranes - a more
complex and challenging approach to incorporate into an inexpensive home or ward
test.
r, if the combined profile of the four classes of markers in sputum does
correlate with lung function performance as we anticipate, we believe we have a
simple screening logy that would allow patients to t an exacerbation. to
better manage their condition at home than can be achieved at present and would
give healthcare providers a tool with which to make earlier interventions.
A further advantage of embodiments of the present invention is that it would allow
clinicians to be able to monitor the effectiveness of the antibiotic treatment within 2-
3 day of administration — far faster than current practice. If the bacterial load is
decreased following treatment, the antibiotic must be effective and vice versa.
Clinical evidence indicates that even when an antibiotic is bringing the infection
under control, it may be some days before the patient actually reports that they feel
well The sense of "wellness" may not correlate closely with the performance of the
antibiotic being administered in the first and critical days after administration (Figure
5).
Perhaps the key benefit of our invention for the ng clinician is the ability to
objectively monitor the performance of the selected otic, by following the
change in concentration of the biomarkers. If the levels of Exotoxin A and
Pyoverdin/Pyocyanin/ferrioxidase do NOT fall, this indicates that the antibiotic is not
effective. Rather than waiting 7 days to see whether the patient recovers, the
Clinician may decide to change to a second otic after just 2 days and so on,
until an ive strategy is arrived at. Potentially, this could reduce a 21-day
process to a 7-day process, reducing inflammation and y saving lung tissue,
3O while reducing hospital admissions and al bed occupation (Figure 6).
Although the present invention may be performed as separate assays on separate
sputum samples, it would be more convenient for the user to have the 3 tests
performed together on a single "dipstick-type" test, for example.
A design concept for such a single test is shown in Figure 7. The test may
orate either a biosensor or lateral flow device. Either approach will require a
able electronics element to quantitate the signal generated
Experimental data
As a proof of concept of the above, we profiled sputum samples from 5 different
patients. For patients colonised with P. nosa, an exacerbation of infection was
detected by profiling the level of Exotoxin A in sputum 7 or more days before the
patient felt so unwell that they presented themselves to the clinic. We have
demonstrated that efficacy of treatment with one antimicrobial compared with
another, can be determined through longitudinal profiling of Exotoxin A in sputum.
We identified two possible fluorescent biomarkers present in sputum. One was
confidently identified as a siderophore produced as an iron scavenging and/or
quorum sensing molecule by the bacterium. its production precedes an
exacerbation in one patient profiled. The identity of the second molecule is at
present n. When this second molecule was present in high trations,
the siderophores produced by the bacteria were not detectable. The presence of
these two markers appears to be ly exclusive. We speculate that this is a
compound (or compounds) ed by the host as it was present in the sputa of
CF ts who were both positive and negative for P. aeruginosa.
We profiled two additional markers for inflammation produced by the host - the
cytokines lL8 and TNFa. While useful, inflammatory markers did not appear to be
good predictors of an exacerbation - itself highly valuable information - but their
levels did show a marked decrease during successful crobial therapy.
Daily sputum samples were ed from five patients.
Five biomarkers were selected which had the highest chance of being useful in
acting as surrogates for the virulence of a colonising pathogen. Virulence is a
ation of the amount of bacteria present (the load, not the tration) and
its activity in sifu in the lung. We selected as kers molecules which play key
roles the bacterium's ability to thrive and multiply or are molecules ed by the
host (the patient) in response to infection or as part of their defence against
infection. We were therefore attempting to create "snap—shot pictures" of the
situation when the sample was taken.
The biomarkers are listed in table 2 below:
None
Iron ging
Haem mono oxygenase
Biliverdin/bilirubin
Inflammatory response:
Cytokine TNF-a
Lactoferrin
The precedence for the use of ial toxins as markers for ion is well
established, e.g., C. difficile Toxins A and B. The weakness of using any one of
these by themselves as sole s in the detection of C. difficile has now been
recognised: these rapid POC tests offer an 80-85% accuracy of detection compared
with cell culture (which at 78 hours is too slow). Some samples simply do not
contain Toxins N8 in detectable amounts and this limits the accuracy of these tests.
By contrast, the use of siderophores (molecules produced by the bacterium which
are involved in cell signalling and in scavenging the iron, and so are essential for
rapid growth) has been reported as a possible biomarker - with mixed results. Not
all of the isolates of P. aeruginosa from the sputum of CF ts, produce this
fluorescent molecule. Our theory is that as the colonising bacteria mutates over the
life of the host, it adapts. As the patient ages and s ever greater lung damage
and has r bleeding into the lungs, the need for the bacterium to expend
energy in ging iron is reduced, and non siderophores—producing clones have
a selective advantage. This may explain why some patients have bacterial colonies
which lack the ability to produce siderophores — they do not need to as it is
metabolically expensive for the bacteria to produce it. So siderophores by
lves are not reliable markers for all patients either. Therefore an approach
which exploits a combination of markers of very different processes undertaken by
the bacteria is useful and gives greater opportunities to capture an accurate
snapshot, irrespective of the state of mutation of the al wild-type bacterium
which colonised the patient in their mid teens.
To protect our lungs from invading pathogens, mammals have evolved mechanisms
to remove iron from the mucus which lines the walls of the lung tissue d to
air. These processes (and there are many) breakdown haemoglobin from blood
which may leak into the fluids covering the lung surfaces, into Iron (II) from the Iron
(Ill) form which the ia require for rapid growth. This makes the iron
unavailable because bacteria cannot uptake iron in the Iron (ll) form. The
breakdown byproducts, catalysed by many enzymes but notably Haem mono
oxygenase, result in the production of coloured ts biliverdin and bilirubin -
most usually seen in our bruises. Following the tion of these ts or the
activity of the host enzyme which catalyses this process and therefore works to
defend us through iron scavenging, is another marker.
Finally, we selected cytokines - small molecules produced by the host as part of an
inflammatory response to invading pathogens and their toxins. Cytokines |L8 and
TN Fa have been detected in the sputum of CF patients by other workers.
Assay development
All of the assays required similar sample preparation to remove the interfering
mucus. We explored several methods to remove this non-homogeneous material,
including mechanical breakdown, chemical digestion and tion by
ultracentrifugation. A ation of chemical digestion and homogenisation worked
well for immunoassay—based tests. This step plus an additional precipitation step of
organic molecules (DNA, fats and proteins) was required for the siderophore assay.
|L8 and TNFa: Assay used was a commercial kit from Millipore using the Luminex
immunoassay bead logy. All samples were profiled for these two biomarkers.
Exotoxin A: in our final Exotoxin A assay. whole sputum was ally ed
and tested "raw" with an immunoassay. We were able to profile ts and
demonstrate that Exotoxin A can be used as a marker to predict exacerbation and
follow the control of infection after the initiation of antimicrobial therapies.
Siderophores. Several compounds have been identified and classified as
siderophores and have a variety of names including: pyocyanin green),
fiuorescein (yellow-green and fluorescent, now also known as pyoverdin), and
pyorubin (red-brown). These have characteristic absorption and scence
a. Previous reports in the literature of the determination of the levels of these
in sputum, involved the use of complex chromatographic clean-up processes and /
or chemical labelling. These are too expensive for large-scale studies. We
developed a very simple method in which these molecules were separated from the
mucus, proteins and DNA using a simple single step precipitation with small
volumes of solvent which was then removed by ation. This simple process
could be readily ted. The precipitation step removed the green colouration
often associated with samples from infected patients and so the resulting pigments
we measured were not proteins such as haemoglobin or alginate, but they were
readily soluble in water and organic solvents. These are likely to be organic
molecules with polar side groups.
Considerable optimisation of the sample preparation process will be possible in
future iterations, but nonetheless and despite these limitations. detection limits for
in A of 0.1 nglml were achieved in whole sputum. Values of 20ng/ml and
above correlated closely with patients becoming ill and requiring treatment with
antimicrobials. Upper values of 160ng/ml in blood have been ed in the
literature. There is considerable scope to improve the limits of detection for this
assay. With our own reagents and fully optimised sample preparation processes
and assays, we anticipate achieving detection limits of 1 nglml in a 10 minute test.
We forecast that this will enable us to detect Exotoxin A before the patient feels
unwell, giving 7-10 days advanced warning of an impending exacerbation.
Resufis
Fluorescence spectra were taken for each of the 260 samples to detect and quantify
the relative concentration of the siderophores population in . Figure 8 shows
the emission spectrum of extracted fraction from patient sputum typical of
siderophores reported in the scientific ture. Excitation was at 300nm and
emission was scanned from 320nm to 500nm. Units ed were Relative
Fluorescent Units (RFUs). This spectrum was from a sputum sample taken from
t 2 towards the end of their profiled period.
We were also surprised to find another entity with a very distinct spectral fingerprint
which had a characteristic peak at 340nm with an excitation of 280nm. The
tration of this material in some samples was so high that they appeared
orange in colouration. Figure 9 shows a characteristic peak at 340nm with an
excitation of 280nm. Note the slight hint of a peak at 410nm. This spectrum was
ed from t 2 at the start of their profiled . This 340 peak
disappeared completely from the sputum of this patient towards the end of their
profiled period.
The identity of peak 340 remains unknown, but the spectrum is characteristic of
haem own products and suggests that patients with high concentrations of
this material are bleeding into their lungs. That this is NOT a product from the
pathogen was confirmed when sputum samples from ill CF patients who were
negative for P. aeruginosa were analysed and found to also n this peak.
Biomarker profiles in 5 CF patients.
We profiled the sputa of 5 patients. Our volunteers were encouraged to give a
sample every day; however there were periods during which samples were not
collected and so there are gaps in the data.
Values of each biomarker were plotted on charts set to the same scale, to make
comparisons between patients easier to make. These plots were then annotated
with patient history, recording wellness or s s, treatment with oral and IV
antimicrobials and admission to clinic.
|L8 and TNFa: the levels expressed varied considerably. in l, levels
increased during an rbation, but were not predictive of an impending
exacerbation. Notably levels of both cytokines decreased during treatment with
antimicrobials, especially when the patient was treated in-clinic.
Exotoxin A: two periods for different patients demonstrate proof-of-concept in
solving the core problems.
Peak 460: additional biomarker to support Exotoxin A, especially for those enjoying
good health for longer periods. Observed in one patient who was well during the
profiled .
Peak 340: useful in assessing the wellness (vulnerability) of patients to
exacerbation.
3O Problem 1 : Early warning of exacerbation.
in A as marker. Patient 5 showed elevated levels of Exotoxin A but ed
as well during routine clinic visit. 14 days later Patient 5 reported as ill and was
admitted. Figure 10 shows this patient's Exotoxin A profile. Values elevated prior to
visit to clinic where they reported as well. Levels continued to rise. 14 days later
Pateint 5 reported as unwell and IV antibiotics were administered. Levels of
in A fall during N treatment.
Siderophore as marker.
Patient 2 showed presence of 340 peak at start of profiling period. These levels fell
to zero with the concomitant se in the 460 peak. The patient ed as ill 5
days later. IV crobials administered 10 days after this. Figure 11 shows the
results from this patient.
Problems 2 & 3: Early decision-making about the effectiveness of an
antimicrobial therapy and objective feedback about the quality of self-
management at home.
Patient 3 provided samples spanning one period of oral antibiotic treatment, three
periods of IV treatment and ion to clinic. There is a wealth of ation
which is summarised in Figure 12.
Exotoxin A: Patient 3 became unwell on the 17th November and began oral
antimicrobials on the 19th November (see Figure 13). Treatment was changed to IV
antimicrobials on the 25th November. Levels of Exotoxin A continued to rise while
on IV treatment at home. Patient 3 reported "feeling better but not right" and was
admitted on the 9th December. Once In clinic, treatment was changed to an
alternative antimicrobial. Levels of Exotoxin A decreased to baseline immediately.
Peaks 340 and 460: Patient 3 showed elevated levels of the 340 peak throughout.
No 460 peak was detected. This was a common theme for all patients with the
exception of Patient 2. With the exception of Patient 2 (21 year old male), all were ill
and being treated with antimicrobials during their profile period including t 4
(21 year old female twin of Patient 2). These patients were selected for this study
because they are regularly ill. We associated high levels of peak 340 over a long
on with patients being ill - perhaps at risk of exacerbation or prone to
bation. Patient 3 fits this hypothesis.
Conclusions from profiling.
Longitudinal ing of sputum from CF ts - an easily available body fluid and
therefore more likely to enjoy patient compliance to a testing regime - can solve
Problems 1 2 and 3 (unmet needs):
- Exotoxin A profiling can be used to predict exacerbations by 7+ days as
demonstrated by patients 3 and 5.
- Siderophores cannot be detected in all patients, especially those consistently ill.
But for those who are well (and therefore not showing peak 340), it could be a
useful co-marker to Exotoxin A (Patient 2)
~ Peak 340 and peak 460 are mutually exclusive (Patient 2).
- Younger patients (2 and 4 who are twins) have similar (and low) concentrations
of peak 340, while the other older patients have high levels which remain
consistently high hout. We associate tent levels of peak 340 with
regular illnesses.
- in A can be used as a marker for the efficacy of antimicrobial therapies.
The e of Patient 3 in which a change in treatment brought about
overnight reduction in levels is compelling.
- Cytokine levels fall during treatment but may show a too generalised response
to be used in profiling for the purpose of solving problems 1 -3.
In conclusion, an ideal test would be a combination of lateral flow s, one for
Exotoxin A, one for peak 340 compound(s) and one for siderophores (peak 460
compounds). Patients with consistently high 340 peak compounds would be
deemed to be vulnerable to bation and monitored closely. Any se in
these patients' Exotoxin A levels would trigger the immediate administration of oral
antimicrobials (Day 1 ) and twice daily g. Failure to reduce levels would trigger
the administration of IV antimicrobials at home (Day 3). Failure to reduce levels
would instigate treatment in clinic, perhaps with an alternative antimicrobial (Day 5).
Once in clinic, Exotoxin A tests would confirm the efficacy of the selected treatment
and would provide reassurance that the infection was under control, giving
confidence for the early release of the patient to home care. On-going treatment at
home would be monitored and any relapse (due to poor self administration of
tion, for example) would be signalled by increased Exotoxin A levels.
Claims (17)
1. A method of determining a level of activity of Pseudomonas aeruginosa bacteria in the lung of a patient, the method comprising: making a first measurement at a first time of a level of at least one marker of a bacterial iron scavenging process and of at least one secreted bacterial protein in a sample of sputum from the lung; making a second measurement at a second time of the levels of said marker and said protein in a sample of sputum from the lung; and determining said level of bacterial activity from changes in said ed levels of said marker of a bacterial iron scavenging process and said secreted bacterial protein over time, n an increase in levels of either said marker or said protein, or both, are indicative of an increase in bacterial activity.
2. A method as claimed in claim 1 sing measuring the level of a plurality of said markers of bacterial iron scavenging processes and determining said level of bacterial activity from said plurality of measured levels.
3. A method as claimed in claim 1 or claim 2 n said marker(s) comprise siderophore(s).
4. A method as claimed in claim 3, wherein said siderophore is one or more of: pyochelin, pyoverdin, pyocyanin.
5. A method as claimed in any one of claims 1, 2, 3 or 4 r comprising measuring the level of at least one iron (Ill) sequestration intermediate for determining said level of bacterial activity.
6. A method as claimed in any one of claims 1 to 5 wherein said protein is a toxin.
7. A method as claimed in claim 6, n said toxin is in A.
8. A method as claimed in any one of claims 1 to 7 wherein said marker of a bacterial iron scavenging process is a siderophore.
9. A method as claimed in any one of claims 1 to 8 wherein said marker is pyoverdin and said protein is exotoxin A.
10. A method of predicting an exacerbation of a level of bacterial ty in the lung of a t, the method comprising making a time series of measurements of bacterial activity on said t using a method as claimed in any one of claims 1 to 9; and using said time series of measurements to predict said exacerbation, wherein an increase in measured activity over the time series is indicative of an exacerbation.
11. A method of determining the effectiveness of an antibiotic treatment of a bacterial lung infection. the method sing making a time series of measurements of ial activity on a patient undergoing said treatment of a lung infection using the method of any one of claims 1 to 9 and determining said effectiveness from a time e of said level of bacterial activity, wherein a decrease in measured activity over the time series is indicative of effectiveness of the ent.
12. A method as d in claim 11 comprising determining that said antibiotic treatment is ineffective if said level of bacterial activity does not fall with time.
13. A method as recited in any one of the preceding claims, wherein said marker has a concentration dependent on a level of quorum signalling between said P. aeruginosa bacteria.
14. A method as recited in any one of the preceding claims, further comprising the step of detecting a host marker of an iron scavenging process.
15. A method as recited in any one of the ing claims, further comprising measuring the level of an inflammatory response of said patient.
16. A method as recited in any one of the preceding claims, wherein the combination of a bacterial toxin, a bacterial marker of iron scavenging process, and a host marker of an iron scavenging process are detected.
17. The method of claim 16, wherein exotoxin A, a siderophore, and haem or a haem breakdown product are detected.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1116234.4 | 2011-09-20 | ||
GBGB1116234.4A GB201116234D0 (en) | 2011-09-20 | 2011-09-20 | Biomarkers for respiratory infection |
GB1213025.8A GB2494953B (en) | 2011-09-20 | 2012-07-23 | Biomarkers for respiratory infection |
GB1213025.8 | 2012-07-23 | ||
PCT/GB2012/052307 WO2013041854A1 (en) | 2011-09-20 | 2012-09-19 | Biomarkers for respiratory infection |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ623074A NZ623074A (en) | 2015-05-29 |
NZ623074B2 true NZ623074B2 (en) | 2015-09-01 |
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