U.S. patent application number 12/671702 was filed with the patent office on 2011-06-09 for use of procalcitonin (pct) in risk stratification and prognosis of patients with a primary, non-infectious disease.
Invention is credited to Andreas Bergmann, Joachim Struck.
Application Number | 20110136161 12/671702 |
Document ID | / |
Family ID | 39745280 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136161 |
Kind Code |
A1 |
Struck; Joachim ; et
al. |
June 9, 2011 |
USE OF PROCALCITONIN (PCT) IN RISK STRATIFICATION AND PROGNOSIS OF
PATIENTS WITH A PRIMARY, NON-INFECTIOUS DISEASE
Abstract
Subject of the present invention are assays and in vitro methods
for the in vitro diagnosis, prognosis and risk stratification of a
patient having a primary, non-infectious disease, whereby the level
of Procalcitonin (PCT) in a sample of a body fluid of the patient
is indicative for the risk of the patient to contract a further
disease or medical condition.
Inventors: |
Struck; Joachim; (Berlin,
DE) ; Bergmann; Andreas; (Berlin, DE) |
Family ID: |
39745280 |
Appl. No.: |
12/671702 |
Filed: |
August 1, 2008 |
PCT Filed: |
August 1, 2008 |
PCT NO: |
PCT/EP08/60176 |
371 Date: |
February 25, 2011 |
Current U.S.
Class: |
435/28 ;
436/86 |
Current CPC
Class: |
A61P 31/02 20180101;
A61K 31/00 20130101; A61P 31/04 20180101; G01N 2333/585 20130101;
G01N 33/74 20130101; G01N 33/6893 20130101 |
Class at
Publication: |
435/28 ;
436/86 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C12Q 1/28 20060101 C12Q001/28 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2007 |
EP |
07015271.5 |
Mar 12, 2008 |
EP |
08152651.9 |
Claims
1. An in vitro method for prognosis for a patient having a primary
disease not being an infection, the method comprising: determining
the level of procalcitonin or fragments thereof of at least 12
amino acids in length, in a sample selected from the group
consisting of a blood sample, a serum sample and a plasma sample or
an extract of any of the aforementioned samples obtained from said
patient; and correlating said level of procalcitonin or fragments
thereof to a risk of the patient to contract a further disease or
medical condition which has not yet been manifested and/or is not
yet symptomatic, wherein said correlating step comprises comparing
said level of procalcitonin or fragments thereof to a threshold
level, whereby, when said level of procalcitonin or fragments
thereof exceeds said threshold level, said patient is predisposed
to said risk and wherein said threshold level is between 0.02 and
0.25 ng/mL.
2. In vitro method according to claim 1, wherein said level of
procalcitonin or fragments thereof is indicative for a bacterial
infection in the patient.
3. In vitro method according to claim 2, wherein said infection is
a local infection.
4. In vitro method according to claim 2, wherein said bacterial
infection is treatable with an antibiotic.
5. In vitro method according to claim 4, wherein the risk of
contracting a further disease or medical condition decreases when
said patient is treated with an antibiotic.
6.-7. (canceled)
8. In vitro method according to claim 1, wherein said primary
disease is selected from the group consisting of cancer, diabetes,
chronic gastrointestinal diseases, chronic renal diseases,
hypertension, orthopaedic diseases including osteoporosis and
neurodegenerative diseases including Alzheimer's disease.
9. In vitro method according to claim 1, wherein said further
disease or medical condition is selected from the group consisting
of cardiological diseases selected from the group consisting of
atherosclerosis, acute coronary syndromes and heart failure.
10. (canceled)
11. A method according to claim 1, further comprising correlating
said level of procalcitonin or fragments thereof with the level of
one or more additional prognostic biomarkers, whereby the
combination of said level of procalcitonin or fragments thereof
with said level of additional prognostic biomarker(s) increases the
predictive value of said level of procalcitonin or fragments
thereof or the level of said related marker for said risk.
12. A method according to claim 11, wherein one of said prognostic
biomarker(s) is proBNP or fragments thereof in a sample obtained
from said patient.
13. A method according to claim 12, wherein said fragment of proBNP
is NT proBNP or BNP.
14. A method according to claim 11, further comprising determining
the level of one or more additional prognostic biomarkers in a
sample obtained from said patient, and correlating both said level
of procalcitonin or fragments thereof and said level of one or more
additional prognostic biomarkers to said predisposition to a risk,
whereby the combination of said level of procalcitonin or fragments
thereof with said level of one or more additional prognostic
biomarkers increases the predictive value of said level of
procalcitonin or fragments thereof for said risk.
15. A method according to claim 11, wherein the additional
prognostic biomarker is selected from a group consisting of
troponin, myeloperoxidase, CRP, neopterin, GDF-15, ST2, cystatin-C,
as well as the following peptides in form of their mature peptides,
precursors, pro-hormones and associated prohormone fragments:
natriuretic peptides, adrenomedullin, endotlielins,
vasopressin.
16. A method according to claim 11, wherein the correlation between
said level of procalciton or fragments thereof and said level of
one or more additional prognostic biomarkers is conducted with a
mathematical algorithm.
17.-27. (canceled)
28. In vitro method according to claim 3, wherein said bacterial
infection is treatable with an antibiotic.
29. In vitro method according to claim 28, wherein the risk of
contracting a further disease or medical condition decreases when
said patient is treated with an antibiotic.
30. In vitro method according to claim 2, wherein said primary
disease is selected from the group consisting of cancer, diabetes,
chronic gastrointestinal diseases, chronic renal diseases,
hypertension, orthopaedic diseases including osteoporosis and
neurodegenerative diseases including Alzheimer's disease.
31. In vitro method according to claim 2, wherein said further
disease or medical condition is selected from the group consisting
of cardiological diseases selected from the group consisting of
atherosclerosis, acute coronary syndromes and heart failure.
32. A method according to claim 2, further comprising correlating
said level of procalcitonin or fragments thereof with the level of
one or more additional prognostic biomarkers, whereby the
combination of said level of procalcitonin or fragments thereof
with said level of additional prognostic biomarker(s) increases the
predictive value of said level of procalcitonin or fragments
thereof or the level of said related marker for said risk.
33. A method according to claim 12, further comprising determining
the level of one or more additional prognostic biomarkers in a
sample obtained from said patient, and correlating both said level
of procalcitonin or fragments thereof and said level of one or more
additional prognostic biomarkers to said predisposition to a risk,
whereby the combination of said level of procalcitonin or fragments
thereof with said level of one or more additional prognostic
biomarkers increases the predictive value of said level of
procalcitonin or fragments thereof for said risk.
34. A method according to claim 13, further comprising determining
the level of one or more additional prognostic biomarkers in a
sample obtained from said patient, and correlating both said level
of procalcitonin or fragments thereof and said level of one or more
additional prognostic biomarkers to said predisposition to a risk,
whereby the combination of said level of procalcitonin or fragments
thereof with said level of one or more additional prognostic
biomarkers increases the predictive value of said level of
procalcitonin or fragments thereof for said risk.
35. A method according to claim 12, wherein the additional
prognostic biomarker is selected from a group consisting of
troponin, myeloperoxidase, CRP, neopterin, GDF-15, ST2, cystatin-C,
as well as the following peptides in form of their mature peptides,
precursors, pro-hormones and associated prohormone fragments:
natriuretic peptides, adrenomedullin, endotlielins,
vasopressin.
36. A method according to claim 13, wherein the additional
prognostic biomarker is selected from a group consisting of
troponin, myeloperoxidase, CRP, neopterin, GDF-15, ST2, cystatin-C,
as well as the following peptides in form of their mature peptides,
precursors, pro-hormones and associated prohormone fragments:
natriuretic peptides, adrenomedullin, endotlielins,
vasopressin.
37. A method according to claim 14, wherein the additional
prognostic biomarker is selected from a group consisting of
troponin, myeloperoxidase, CRP, neopterin, GDF-15, ST2, cystatin-C,
as well as the following peptides in form of their mature peptides,
precursors, pro-hormones and associated prohormone fragments:
natriuretic peptides, adrenomedullin, endotlielins, vasopressin.
Description
[0001] Subject of the present invention is the in vitro diagnosis,
prognosis and risk stratification of a patient having a primary,
non-infectious disease, whereby the level of Procalcitonin (PCT) in
a sample of a body fluid of the patient is indicative for the risk
of the patient to contract a further disease or medical
condition.
BACKGROUND OF THE INVENTION
[0002] Procalcitonin (PCT) has become a well-established biomarker
for sepsis diagnosis: PCT reflects the severity of bacterial
infection and is in particular used to monitor progression of
infection into sepsis, severe sepsis, or septic shock. PCT
concentrations in sepsis, severe sepsis, or septic shock are
typically above 1 ng/mL. It is possible to use PCT to measure the
activity of the infection-associated systemic inflammatory
response, to control success of therapy, and to estimate prognosis
(Assicot M et al.: High serum procalcitonin concentrations in
patients with sepsis and infection. Lancet 1993, 341:515-8; Clec'h
C et al.: Diagnostic and prognostic value of procalcitonin in
patients with septic shock. Crit. Care Med 2004; 32:1166-9; Lee Y J
et al.: Predictive comparisons of procalcitonin (PCT) level,
arterial ketone body ratio (AKBR), APACHE III score and multiple
organ dysfunction score (MODS) in systemic inflammatory response
syndrome (SIRS), Yonsei Med J 2004, 45, 29-37; Meisner Biomarkers
of sepsis: clinically useful? Curr Opin Crit. Care 2005, 11,
473-480; Wunder C et al.: Are IL-6, IL-10 and PCT plasma
concentrations reliable for outcome prediction in severe sepsis? A
comparison with APACHE III and SAPS IL Inflamm Res 2004, 53,
158-163). The increase of PCT levels in patients with sepsis
correlates with mortality (Oberhoffer M et al.: Outcome prediction
by traditional and new biomarkers of inflammation in patients with
sepsis. Clin Chem Lab Med 1999; 37:363-368).
[0003] An increasing number of studies discusses the potential role
of PCT in non-septic infectious diseases like pneumonia, bacterial
meningitis and malaria (Bugden S A et al.: The potential role of
procalcitonin in the emergency department management of febrile
young adults during a sustained meningococcal epidemic. Emerg Med
Australas 2004, 16, 114-119; Chiwakata C B et al.: Procalcitonin as
a parameter of disease severity and risk of mortality in patients
with Plasmodium falciparum malaria. J Infect Dis 2001, 183,
1161-1164; Schwarz S et al.: Serum procalcitonin levels in
bacterial and abacterial meningitis, Crit. Care Med 2000, 28,
1828-1832).
[0004] In vitro-studies showed that PCT plays an important role
during monocyte adhesion and migration and further has an effect on
inducible nitric oxide synthase (iNOS) gene expression (Linscheid P
et al.: Expression and secretion of procalcitonin and calcitonin
gene-related peptide by adherent monocytes and by
macrophage-activated adipocytes, Crit. Care Med 2004, 32,
1715-1721; Wiedermann F J et al.: Migration of human monocytes in
response to procalcitonin, Crit. Care Med, 2002, 30, 1112-1117;
Hoffmann O et al.: Procalcitonin amplifies inducible nitric oxide
synthase gene expression and nitric oxide production in vascular
smooth muscle cells, Crit. Care Med, 2002, 30, 2091-2095.).
[0005] PCT has been used to guide antibiotic therapy (Christ-Crain
M et al.: Effect of procalcitonin-guided treatment on antibiotic
use and outcome in lower respiratory tract infections:
cluster-randomised, single-blinded intervention trial, Lancet, 2004
Feb. 21; 363(9409):600-7): In patients with symptoms of lower
respiratory tract infections presenting at the emergency department
PCT was measured, and only patients with PCT concentrations
>0.25 ng/mL or >0.5 ng/mL were treated with antibiotics,
Apparently, this regimen led to a clinical outcome
undistinguishable from the control group, in which also many
patients with PCT concentrations <0.25 ng/mL received
antibiotics. Of note, in this study, patients with relevant
comorbidities as for instance heart failure were excluded. Thus, it
is unclear so far, whether the presence of a primary disease in
addition to an infection should influence the interpretation of PCT
concentrations below 0.25 ng/mL. A relevant primary disease might
put an additional burden on the immune system, and biomarkers of
infection, such as PCT, in such situation might be indicative of an
infection in a different, i.e. lower, concentration range than in
the absence of a relevant primary disease.
[0006] In healthy indivivals, the PCT concentration is well below
0.25 ng/mL: The median concentration has been determined to be
0.014 ng/mL (Morgenthaler N O et al.: Sensitive immunoluminometric
assay for the detection of procalcitonin. Clin Chem. 2002 May;
48(5):788-90.)
[0007] A method for diagnosis of infections or inflammatory
diseases of the airways and lungs with associated heart failure,
wherein the biomarker procalcitonin is determined in a patient, is
described in WO 2008/040328 A2.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the surprising finding
that in samples of patients with a primary, non-infectious disease,
slightly elevated procalcitonin (PCT) levels (concentrations) have
been detected at a large frequency and are of diagnostic relevance.
Remarkably, the inventors have identified a large number of samples
having serum levels above 0.03 ng/mL (26.0%) and 0.05 ng/mL
(14.7%), respectively, from a total of 4997 samples of patients
having a primary, non-infectious disease. Slightly elevated PCT
levels relate to PCT levels in the range of from about 0.02 to 0.25
ng/mL, preferably between about 0.02 and 0.1 ng/mL. The presence of
slightly elevated PCT levels may be indicative for the risk of a
patient having a non-infectious primary disease to acquire a yet
clinically unmanifested and/or yet asymptomatic further disease or
medical condition. Such a further disease or medical condition may
be related to a local infection or the local infection may
facilitate, accelerate and/or enhance the risk of contracting or
acquiring the further disease or medical condition. The present
invention provides a method for the prognosis of a patient's risk
to acquire a further disease or medical condition in addition to an
non-infectious primary disease. This also allows for adaption of
the treatment of these patients, e.g. by an additional antibiotics
therapy which the patient would not have necessarily received if
the elevated PCT level had not been detected. It has to be noted
that so far, patients with non-infectious primary diseases are not
routinely screened for their PCT levels during diagnosis. It is
taught by the present invention that patients with a primary
disease not being an infection should be assayed for their PCT
level to allow for the prognosis of the risk to acquire a further
disease or medical condition and ultimately to allow for the
adaption of therapy.
[0009] Hence, the present invention provides an in vitro method for
prognosis for a patient having a primary disease not being an
infection, the method comprising: determining the level of
procalcitonin or fragments thereof of at least 12 amino acids in
length, preferably more than 50 amino acids in length, more
preferably more than 110 amino acids in length, in a sample
obtained from said patient; and correlating said level of
procalcitonin or fragments thereof to a risk of the patient to
contract a further disease or medical condition which has not yet
been manifested and/or is not yet symptomatic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows a histogram plot of the frequency of
procalcitonin level ranges in patients' samples. These were 4997
unselected samples consecutively sent by physicians of various
specialties to a private laboratory for various types of analysis,
but not PCT.
[0011] FIG. 2 shows the distribution of PCT levels above 0.05 ng/mL
in relation the medical field of the consulting specialist
physician who provided the respective sample. Medians in all groups
are indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention relates to an in vitro method for
prognosis for a patient having a primary disease not being an
infection, the method comprising: determining the level of
procalcitonin or fragments thereof of at least 12 amino acids in
length, preferably more than 50 amino acids in length, more
preferably more than 110 amino acids in length, in a sample
obtained from said patient; and correlating said level of
procalcitonin or fragments thereof to a risk of the patient to
contract a further disease or medical condition which has not yet
been manifested and/or is not yet symptomatic.
[0013] "Patients" in the meaning of the invention are understood to
be all persons or animals, irrespective whether or not they exhibit
pathological changes, unless stated otherwise. In a preferred
embodiment the patient according to the invention is a human.
[0014] Preferably, the patient in the context of the present
invention has a primary disease not being an infection and the
level of procalcitonin or fragments thereof of at least 12 amino
acids in length, in serum or plasma samples of said patient is
below 0.25 ng/mL.
[0015] In the present invention, the term "prognosis" denotes a
prediction of how a subject's (e.g. a patient's) medical condition
will progress. This may include an estimation of the chance of
recovery or the chance of an adverse outcome for said subject.
[0016] As mentioned herein in the context of proteins or peptides,
the term "fragment" refers to smaller proteins or peptides
derivable from larger proteins or peptides, which hence comprise a
partial sequence of the larger protein or peptide. Said fragments
are derivable from the larger proteins or peptides by
saponification of one or more of its peptide bonds.
[0017] Procalcitonin is a 116 amino acids comprising peptide.
Smaller variants, such as for instance PCT 3-116 and others, exist
as well. Thus the length of procalcitonin fragments is at least 12
amino acids, preferably more than 50 amino acids, more preferably
more than 110 amino acids.
[0018] Preferably herein, said risk of contracting a further
disease or medical condition is related to an existing bacterial
infection, particularly a local infection. A local infection may
facilitate, accelerate and/or enhance the risk of contracting or
acquiring a further disease or medical condition in a patient
having a non-infectious primary disease.
[0019] In particularly preferred embodiments of the in vitro
method, said level of procalcitonin or fragments thereof is
indicative for a bacterial infection in the patient.
[0020] It is further preferred that said infection is a local
infection.
[0021] A local infection herein relates to all infections being
less severe than a sepsis. A sepsis is defined as an infection
being associated with the "Systemic Inflammatory Response Syndrome"
("SIRS") (Levy M M et al. 2001 SCCM/ESICM/ACCP/ATS/SIS
International Sepsis Definitions Conference. Crit. Care Med. 2003
April; 31(4):1250-6).
[0022] The local infection may for example be an infection in the
oral cavity, at the teeth or the root of the teeth, infection in
wounds, infection in the respiratory tract, or haemorrhoids, or
others.
[0023] Said local infection herein may be treated by administration
of an antibiotic. Treatment of the local infection leads to a
decreased risk of the patient for acquiring the further disease or
medical condition. Hence, in a preferred embodiment of the in vitro
method, said bacterial infection is treatable with an antibiotic.
In this case it is preferred, that the risk of contracting a
further disease or medical condition decreases when the patient is
treated with an antibiotic.
[0024] The correlating step of the in vitro method of the present
invention preferably comprises comparing said level of
procalcitonin or fragments thereof to a threshold level, whereby,
when said level of procalcitonin or fragments thereof exceeds said
threshold level, said patient is predisposed to said risk.
[0025] Said threshold level is preferably between 0.02 and 0.25
ng/mL, more preferably between 0.02 and 0.1 ng/mL, even more
preferably at about 0.05 (+/-0.01) ng/mL, and most preferably at
about 0.03 (+1-0.01) ng/mL. Definition of these threshold levels
comes from the analysis of the frequency distribution of PCT level
ranges shown in the histogram of appended FIG. 1 and Example 1.
[0026] In a particular embodiment said primary disease is not
arteriosclerosis. In another embodiment said primary disease is not
heart failure. It is in some embodiments of the invention said
primary disease is not an acute coronary syndrome. Furthermore, it
is in a particular embodiment preferred that said primary disease
is not a coronary disease. In a particular embodiment the further
disease or medical condition is not selected from the group
comprising acute coronary syndrome, heart failure or myocardial
infarction.
[0027] In one embodiment said further disease or medical condition
is not an infection, particularly not an infection of the airways
and lungs.
[0028] In another embodiment of the in vitro method according to
the invention, said primary disease is selected from but not
restricted to the group comprising cancer, diabetes, chronic
gastrointestinal diseases, chronic renal diseases, hypertension,
orthopaedic diseases including osteoporosis, and neurodegenerative
diseases including Alzheimer's disease.
[0029] Furthermore, in another embodiment said further disease or
medical condition is selected from the group comprising
cardiological diseases selected from but not restricted to the
group comprising atherosclerosis, acute coronary syndromes and
heart failure.
[0030] The primary disease herein relates to a disease which is
already manifested and/or is already symptomatic. The further
disease or medical condition relates to a disease which is not yet
manifested and/or is not yet symptomatic.
[0031] The sample from the patient may for example be selected from
the group comprising a blood sample, a serum sample, a plasma
sample, and an urine sample or an extract of any of the
aforementioned samples.
[0032] In a particular embodiment of the invention, the in vitro
method further comprises mathematically combining said level of
procalcitonin or fragments thereof with the level of one or more
additional prognostic biomarkers, whereby the combination of said
level of procalcitonin or fragments thereof with said level of
additional prognostic biomarker(s) increases the predictive value
of said level of procalcitonin or fragments thereof or the level of
said related biomarker for said risk of contracting a further
disease or medical condition.
[0033] In the context of the present invention, an "algorithm" or
"mathematical algorithm" refers to the use of a mathematical or
statistical method or model used to compare a certain measured
value with values of a reference population in order to stratify
said measured value. This may for instance be the median of the
level of a certain entity in an ensemble of pre-determined samples,
which means that the measured level of said entity is compared with
the mathematical median of the level of said entity in a given
number of samples. The number of samples used to determine the
median is not particularly limited, but should be sufficient in
order to ensure statistical significance of the median. The number
of samples used to determine the median may even increase over the
course of time, as the results of further measurement values from
clinical samples are added in order to increase the statistic
significance of the median. Preferably, the sample number is chosen
such that statistical significance of the median is ensured. Thus,
said median is used as a reference value, whereby the measured
level of the aforementioned entity can be statistically correlated
with a certain physiological state, e.g. the propensity of an
adverse outcome for a patient, depending on the relative level
above or below the median and the extent of deviation of the
measured value from said median. In place of the median, other
statistical methods, such as the determination of quantiles (e.g.
quartiles or percentiles) or mathematical models, preferably Cox
Regression may be used analogously to the above description in
order to obtain the above-mentioned reference value and/or
otherwise determine the significance of a measured value with
respect to the physiological status of a given subject from which
the sample has been obtained. Said mathematical or statistical
methods or models are well known to the person skilled in the art
and the use thereof in the context of medicinal applications is
well established.
[0034] The "term" biomarker (biological marker) relates to
measurable and quantifiable biological parameters (e.g., specific
enzyme concentration, specific hormone concentration, specific gene
phenotype distribution in a population, presence of biological
substances) which serve as indices for health- and
physiology-related assessments, such as disease risk, psychiatric
disorders, environmental exposure and its effects, disease
diagnosis, metabolic processes, substance abuse, pregnancy, cell
line development, epidemiologic studies, etc. Furthermore, a
biomarker is defined as a characteristic that is objectively
measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention. A biomarker may be measured on a
biosample (as a blood, urine, or tissue test), it may be a
recording obtained from a person (blood pressure, ECG, or Holter),
or it may be an imaging test (echocardiogram or CT scan) (Vasan et
al. 2006, Circulation 113:2335-2362).
[0035] Biomarkers can indicate a variety of health or disease
characteristics, including the level or type of exposure to an
environmental factor, genetic susceptibility, genetic responses to
exposures, biomarkers of subclinical or clinical disease, or
indicators of response to therapy. Thus, a simplistic way to think
of biomarkers is as indicators of disease trait (risk factor or
risk biomarker), disease state (preclinical or clinical), or
disease rate (progression). Accordingly, biomarkers can be
classified as antecedent biomarkers (identifying the risk of
developing an illness), screening biomarkers (screening for
subclinical disease), diagnostic biomarkers (recognizing overt
disease), staging biomarkers (categorizing disease severity), or
prognostic biomarkers (predicting future disease course, including
recurrence and response to therapy, and monitoring efficacy of
therapy). Biomarkers may also serve as surrogate end points. A
surrogate end point is one that can be used as an outcome in
clinical trials to evaluate safety and effectiveness of therapies
in lieu of measurement of the true outcome of interest. The
underlying principle is that alterations in the surrogate end point
track closely with changes in the outcome of interest. Surrogate
end points have the advantage that they may be gathered in a
shorter time frame and with less expense than end points such as
morbidity and mortality, which require large clinical trials for
evaluation. Additional values of surrogate end points include the
fact that they are closer to the exposure/intervention of interest
and may be easier to relate causally than more distant clinical
events. An important disadvantage of surrogate end points is that
if clinical outcome of interest is influenced by numerous factors
(in addition to the surrogate end point), residual confounding may
reduce the validity of the surrogate end point. It has been
suggested that the validity of a surrogate end point is greater if
it can explain at least 50% of the effect of an exposure or
intervention on the outcome of interest. For instance, a biomarker
may be a protein, peptide or a nucleic acid molecule.
[0036] One of said prognostic biomarker(s) is preferably proBNP or
fragments thereof in a sample obtained from said patient. More
preferably, said fragment of proBNP is NT pro-BNP or BNP.
[0037] In a particular embodiment, the in vitro method further
comprises determining the level of one or more additional
prognostic biomarkers in a sample obtained from said patient, and
correlating both said level of procalcitonin or fragments thereof
and said level of one or more additional prognostic biomarkers to
said predisposition to a risk, whereby the combination of said
level of procalcitonin or fragments thereof with said level of one
or more additional prognostic biomarkers increases the predictive
value of said level of procalcitonin or fragments thereof for said
risk.
[0038] The additional prognostic biomarker may for example be
selected from a group comprising troponin, myeloperoxidase, CRP,
neopterin, GDF-15, ST2, cystatin-C, as well as the following
peptides in form of their mature peptides, prohormones (precursors)
and associated prohormone fragments: natriuretic peptides,
adrenomedullin, endothelins, vasopressin.
[0039] Preferably, the correlation between said level of
procalcitonin or fragments thereof and said level of one or more
additional prognostic biomarkers is conducted with a mathematical
algorithm.
[0040] In a further aspect the present invention relates to the use
of an ultrasensitive procalcitonin assay having a lower limit of
detection of below about 0.05 ng/mL, preferably below about 0.04
ng/mL, more preferably below about 0.03 ng/mL, most preferably
below about 0.02 ng/mL for determining in a patient having a
primary disease not being an infection the risk of the patient to
contract a further disease or medical condition which has not yet
been manifested and/or is not yet symptomatic.
[0041] In a preferred embodiment of the use of the ultrasensitive
procalcitonin assay, the level of procalcitonin or fragments
thereof of at least 12 amino acids in length or a mixture of
procalicitonin and/or fragments thereof, is determined in a sample
from said patient. In one embodiment the level of only one fragment
is determined. In another embodiment the level of a mixture of
procalicitonin and/or fragments thereof is determined.
[0042] As mentioned herein, an "assay" or "diagnostic assay" can be
of any type applied in the field of diagnostics, including but not
restricted to assays methods based on enzymatic reactions,
luminescence, fluorescence or radiochemicals. The preferred
detection methods comprise rapid tests (point-of-care tests),
radioimmunoassays, chemiluminescence- and
fluorescence-immunoassays, Immunoblot assays, Enzyme-linked
immunoassays (ELISA), Luminex-based bead arrays, and protein
microarray assays. The assay types can further be microtitre
plate-based, chip-based, bead-based, wherein the biomarkers can be
attached to the surface or in solution. The assays can be
homogenous or heterogeneous assays, sandwich assays, competitive
and non-competitive assays. In a particularly preferred embodiment,
the assay is in the form of a sandwich assay, which is a
noncompetitive immunoassay, wherein the molecule to be detected
and/or quantified is bound to a first antibody and to a second
antibody. The first antibody may be bound to a solid phase, e.g. a
bead, a surface of a well or other container, a chip or a strip,
and the second antibody is an antibody which is labeled, e.g. with
a dye, with a radioisotope, or a reactive or catalytically active
moiety. The amount of labeled antibody on the site is then measured
by an appropriate method. The general composition and procedures
involved with "sandwich assays" are well-established and known to
the skilled person (The Immunoassay Handbook, Ed. David Wild,
Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267;
Hultsehig C et al., Curr Opin Chem. Biol. 2006 February;
10(1):4-10. PMID: 16376134), incorporated herein by reference.
[0043] In a particularly preferred embodiment the assay comprises
two capture molecules (capture probes), preferably antibodies which
are both present as dispersions in a liquid reaction mixture,
wherein a first marking component is attached, to the first capture
molecule, wherein said first marking component is part of a marking
system based on fluorescence- or chemiluminescence-quenching or
amplification, and a second marking component of said marking
system is attached to the second capture molecule, so that upon
binding of both capture molecules to the analyte to be detected, a
measurable signal is generated that allows for the detection of the
formed sandwich complexes in the solution comprising the
sample.
[0044] In the context of the present invention, a capture probe may
be selected from the group comprising a nucleic acid molecule,
particularly an aptamer, a carbohydrate molecule, a PNA molecule, a
protein, an antibody, a peptide, particularly a cyclic peptide or a
glycoprotein.
[0045] Even more preferred, said marking system comprises rare
earth cryptates or rare earth chelates in combination with a
fluorescence dye or chemiluminescence dye, in particular a dye of
the cyanine type.
[0046] In the context of the present invention, fluorescence based
assays comprise the use of dyes, which may for instance be selected
from the group comprising FAM (5- or 6-carboxyfluorescein), VIC,
NED, Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800,
Cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen,
6-Carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET,
6-Carboxy-4',5'-dichloro-2',7'-dimethodyfluorescein (JOE),
N,N,N',N'-Tetramethyl-6-carboxyrhodamine (TAMRA),
6-Carboxy-X-rhodamine (ROX), 5-Carboxyrhodamine-60 (R6G5),
6-carboxyrhodamine-6G (RG6), Rhodamine,Rhodamine Green, Rhodamine
Red, Rhodamine 110, BODIPY dyes, such as BODIPY TMR, Oregon Green,
Coumarines such as Umbelliferone, Benzimides, such as Hoechst
33258; Phenanthridines, such as Texas Red, Yakima Yellow, Alexa
Fluor, PET, Ethidiumbromide, Acridinium dyes, Carbazol dyes,
Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, and the
like.
[0047] In the context of the present invention, chemiluminescence
based assays comprise the use of dyes, based on the physical
principles described for chemiluminescent materials in Kirk-Othmer,
Encyclopedia of chemical technology, 4.sup.th ed., executive
editor, J. L Kroschwitz; editor, M. Howe-Grant, John Wiley &
Sons, 1993, vol. 15, p. 518-562, incorporated herein by reference,
including citations on pages 551-562. Preferred chemiluminescent
dyes are acridiniurn esters.
[0048] In the context of the present invention, the term
"ultrasensitive procalcitonin assay" means that the assay for the
detection of procalcitonin or fragments thereof and/or
quantification of the level thereof has a lower limit of detection
of below about 0.05 ng/mL, preferably below about 0.04 ng/mL, more
preferably below about 0.03 ng/mL, most preferably below about 0.02
ng/mL.
[0049] An ultrasensitive PCT assay is for example the PCT sensitive
LIA (Luminescence immuno Assay) Kit (B.R.A.H.M.S AG, Hennigsdorf,
Germany, Product No. 109.050). PCT levels in the context of the
present invention may for example be determined with an assay as
described above, preferably with the PCT sensitive LIA.
(Luminescence Immuno Assay) Kit (B.R.A.H.M.S AG, Hennigsdorf,
Germany, Product No. 109.050) as in example 1 or following the
general procedure described in example 2.
[0050] The use of an ultrasensitive procalcitonin assay is
preferably for stratifying the risk for contracting a further
disease or medical in a patient having a primary disease.
[0051] The ultrasensitive procalcitonin assay may be used for the
control of the treatment or prevention of said further disease or
medical condition. Preferably said treatment or prevention
comprises administration of an antibiotic to the patient.
[0052] The ultrasensitive procalcitonin assay may for example be a
sandwich assay comprising two antibodies against different moieties
of procalcitonin.
[0053] In a particular embodiment of the use of the ultrasensitive
procalcitonin assay, one antibody is against the calcitonin moiety
of procalcitonin, and the other antibody is a monoclonal antibody
against the katacalcin moiety of procalcitonin.
[0054] In the context of the present invention, the term
"calcitonin moiety of procalcitonin" refers to a polypeptide
comprising the amino acids 85-116 of pre-procalcitonin. In the
context of the present invention, the "katacalcin moiety of
procalcitonin" refers to a polypeptide comprising the amino acids
121-141 of pre-procalcitonin. The above amino acid numbers refer to
the sequence of human pre-procalcitonin as listed in Protein data
bank entry http://www.expasy.ch/uniprot/P01258. Also encompassed
are amino acid sequences of analogous origin analogous in other
species, as well as polypeptides with preferably, at least 90%,
more preferably at lest 95%, most preferably at least 98% sequence
homology to the above-mentioned human polypeptides.
[0055] In yet another aspect the present invention relates to an
antibiotic for the use in the treatment of a local infection for
the prevention of a further disease or medical condition which has
not yet been manifested in a patient having a primary disease,
wherein said primary disease is not an infection and wherein the
antibiotic is administered when the level of procalcitonin or
fragments thereof of at least 12 amino acids in length, in a sample
of the patient selected from the group comprising a blood sample, a
serum sample and a plasma sample, is between 0.02 and 025 ng/mL,
preferably between 0.02 and 0.1 ng/mL. Preferably, the risk of
contracting a further disease or medical condition decreases when
the said patient is treated with an antibiotic.
[0056] In a further aspect the invention relates to an in vitro
method for diagnosis of the presence of a bacterial infection in a
patient, the method comprising: determining the level of
procalcitonin or fragments thereof of at least 12 amino acids in
length, in a sample obtained from said patient:
(i) at least once before the start of said antibiotic treatment or
within six hours after the start of the treatment, and (ii) at
least once after 12 hours to 1 week alter the start of an
antibiotic treatment of the patient; and correlating said level of
procalcitonin or fragments thereof to the presence of a bacterial
infection, wherein a decrease of said level of at least 20% per 24
h ng/mL is indicative for the presence of a bacterial infection in
the patient.
[0057] Preferably, the patient has a primary disease not being an
infection and the level of procalcitonin or fragments thereof of at
least 12 amino acids in length, in serum or plasma samples of said
patient is below 0.25 ng/mL.
[0058] The term "antibiotic" in the context of the present
invention refers to a chemical substance, which has the capacity to
inhibit the growth of or to kill microorganisms. Different
antibiotics may have various mechanism of action, e.g. by binding
to bacterial ribosomal subunits, thereby inhibiting protein
biosynthesis, inhibiting cell wall synthesis, e.g. by inhibiting
peptidoglycan synthesis, interacting with the bacterial cytoplasmic
membrane, thereby e.g. changing its permeability, inhibit bacterial
DNA gyrase or topoisomerase IV enzyme, thereby inhibiting DNA
replication and transcription, inhibiting folate synthesis, or
inhibiting transcription by binding to RNA polymerase. The
antibiotic in the context of the present invention may for example
be selected from the group comprising .beta.-Lactames,
glykopeptides, polyketides, aminoglycoside antibiotics, polypeptide
antibiotics, chinolones and sulfonamides. Preferably, the term
refers to beta-lactam compounds like penicillines, cephalosporins
or carbapenems; tetracyclines; macrolides; fluoroquinolones;
sulphonamides; aminoglycosides; imidazoles; peptide-antibiotics and
lincosamides. More preferably, the term relates to amoxicillin,
flucloxacillin, penicillin G, ampicillin, methicillin, oxacillin,
cefoxitin, ceftriaxone, ceftrizoxime, imipenem, erythromacin,
tylosin, tilmicosin, spiramycin, josamycin, azithromycin,
clarithromycin, tetracycline, minocycline, doxycycline,
lymecycline, norfloxacin, enoxacin, ofloxacin, co-trimoxazole,
ciprofloxacin, trimethoprim, gentamicin, amikacin, metronidazole,
bactiracin, clindamycin or lincomycin. Most preferably, the term
relates to ampicillin, cefotaxime, erythromycin, tetracycline,
ciprofloxacin, co-trimoxazole, gentamicin, metronidazole,
bacitracin or clindomycin.
EXAMPLES
Example 1
Determination of Procalcitonin Levels in Samples of Patients with
Various Primary Diseases
[0059] 4997 consecutive blood sera samples of patients of a
clinical lab have been analyzed to determine the level of
procalcitonin (PCT) using the B.R.A.H.M.S PCT sensitive LIA
(Luminescence Immuno Assay) Kit (B.R.A.H.M.S AG, Hennigsdorf,
Germany, Product No. 109.050). The patients sera have been sent for
analysis to the lab by different consulting specialist physicians
from various medical fields, such as nephrology, urology, oncology,
pediatrics, internal medicine, general medicine and others. The
assay has been performed according to the manual shipped with the
kit, except that the sample volume has been increased form 50 .mu.L
to 100 .mu.L to increase the functional assay sensitivity (FAS) and
to reliably determine the PCT concentrations in the lower
concentration range (0.05 to 0.25 ng/mL).
[0060] The frequencies of the determined PCT levels have been
plotted in a histogram (see appended FIG. 1). 663% of the sera
samples showed PCT levels above 0.017 ng/mL, 26.0% of the sera
samples showed PCT concentrations of above 0.03 ng/mL and 14.0% of
the sera samples showed PCT levels of above 0.05 ng/mL. The samples
having PCT concentrations above 0.05 ng/mL (i.e. 702 samples out of
4997 samples) have been classified according to the medical field
of the consulting specialist physician from which the respective
sample originated. This correlation is plotted in appended FIG. 2,
The high number of patients having a primary disease not being an
infection but nevertheless having PCT levels above 0.03 ng/mL and
0.05 ng/mL, respectively, is a surprising finding.
Example 2
General Procedure for the Determination of Procalcitonin Levels in
Samples of Patients
[0061] Procalcitonin (PCT) can be measured as described
(Morgenthaler N G et al.: Clin Chem, 2002 May, 48(5), 788-790).
Sheep antibodies were raised against the calcitonin moiety of PCT,
and a mouse monoclonal antibody was raised against the katacalcin
moiety of PCT. Tubes were coated with the anti-katacalcin antibody.
The anti-Calcitonin antibody was labelled with MACN Acridiniumester
(InVent GmbH, Hennigsdorf, Germany) and served as tracer, Dilutions
of recombinant PCT in normal horse serum served as standards. 100
.mu.l sample or standard were incubated in the coated tubes for 30
minutes, 200 .mu.l tracer were added. After incubation for 2 h the
tubes were washed 4 times with 1 ml of LIA wash solution
(B.R.A.H.M.S AG), and bound. Chemiluminescence was measured using a
LB952T luminometer (Berthold, Germany).
* * * * *
References