U.S. patent application number 16/138555 was filed with the patent office on 2019-03-14 for diagnostic method for urinary tract infection.
The applicant listed for this patent is Hansa Medical AB. Invention is credited to Per AKESSON, Lars BJORCK, Bertil CHRISTENSSON, Heiko HERWALD, Adam LINDER.
Application Number | 20190079104 16/138555 |
Document ID | / |
Family ID | 43836342 |
Filed Date | 2019-03-14 |
United States Patent
Application |
20190079104 |
Kind Code |
A1 |
BJORCK; Lars ; et
al. |
March 14, 2019 |
DIAGNOSTIC METHOD FOR URINARY TRACT INFECTION
Abstract
It has been demonstrated that the urinary level of HBP increases
in individuals that have a urinary tract infection. Accordingly,
the urinary level of HBP in an individual can be used to determine
whether or not an individual has a urinary tract infection.
Inventors: |
BJORCK; Lars; (Lund, SE)
; CHRISTENSSON; Bertil; (Lund, SE) ; HERWALD;
Heiko; (Veberod, SE) ; LINDER; Adam; (Lund,
SE) ; AKESSON; Per; (Lund, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hansa Medical AB |
Lund |
|
SE |
|
|
Family ID: |
43836342 |
Appl. No.: |
16/138555 |
Filed: |
September 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15153570 |
May 12, 2016 |
10126312 |
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16138555 |
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13983224 |
Nov 19, 2013 |
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PCT/EP2012/052059 |
Feb 7, 2012 |
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15153570 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/33 20130101;
G01N 33/6893 20130101; A61K 45/06 20130101; G01N 2400/40 20130101;
G01N 33/569 20130101; G01N 2800/348 20130101; C07K 16/18 20130101;
A61P 13/02 20180101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 16/18 20060101 C07K016/18; A61K 45/06 20060101
A61K045/06; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2011 |
GB |
1102108.6 |
Claims
1-14. (canceled)
15. A method of identifying whether an individual has a urinary
tract infection (UTI), comprising: measuring the level of Heparin
Binding Protein (HBP, CAP37, Azuricidin) in a urine sample obtained
from an individual by; (i) contacting the sample with an antibody
which binds specifically to HBP; and (ii) quantitating the amount
of antibody-HBP complex to measure the HBP level in the urine
sample; and detecting the presence of UTI in the individual at
about 93% Sensitivity or greater and at about 90% Specificity or
greater based on the measured HBP level in the urine sample.
16. The method of claim 15, wherein the individual is a mammal.
17. The method of claim 16, wherein the mammal is a human.
18. The method of claim 17, wherein the detecting of UTI at about
93% Sensitivity or greater and at about 90% Specificity or greater
is determined by a Receiver Operating Characteristic (ROC).
19. The method of claim 17, wherein the 93.3% Sensitivity and the
90.3% Specificity for detecting UTI is a threshold value of about
32 ng/ml in the urine sample.
20. The method of claim 15, wherein the binding of the antibody to
HBP is measured by enzyme linked immunosorbent assay (ELISA).
21. The method of claim 15, wherein the binding of the antibody to
HBP is measured using a dipstick assay.
22. The method of claim 15, wherein the urinary tract infection is
lower urinary tract infection or pyelonephritis.
23. The method of claim 15, further comprising determining the
white blood cell count or the level of IL6 in a urine sample from
the individual, or conducting the nitrite test on a urine sample
from the individual.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the diagnosis and treatment of
urinary tract infection.
BACKGROUND OF THE INVENTION
[0002] Urinary tract infection (UTI) is a common diagnosis that can
result in serious complications in both children and adults.
Efficient diagnosis and treatment is important to reduce the risk
of renal failure and overuse of antibiotics. Rapid and safe
diagnostic methods are required as an alternative to bacterial
cultures. Early treatment improves prognosis of urinary tract
infection, and therefore early diagnosis is vital. A reliable
biological or clinical marker to determine as early as possible
whether or not an individual has a urinary tract infection is
needed.
SUMMARY OF THE INVENTION
[0003] Heparin-binding protein (HBP, CAP37, Azurocidin) is a
glycosylated, single chain, positively charged 37 kDa inactive
serine protease homologue exhibiting 44% sequence identity with
human neutrophil elastase. The three dimensional structure of HBP
has been published (Iversen et al Nat Struct Biol. 1997 April;
4(4):265-8). It is contained in the azurophilic granulae and
secretory vesicles of human neutrophils (Lindmark et al, J Leukoc
Biol 1999; 66(4):634-43 and Tapper et al, Blood 2000;
96:2329-2337). It is a multifunctional protein that has been shown
to induce vascular leakage by altering the Ca.sup.2+ balance of the
blood vessel cytoskeleton (Gautam et al, Nature Medicine 2001;
7(10):1123-7). The M-protein of group A streptococci (GAS) in
complex with fibrinogen has been shown to induce HBP release by
stimulation of the B2-integrin receptor of neutrophils (Herwald et
al, Cell 2004; 116(3):367-79). LPS can also induce HBP release by
an unknown mechanism (Rasmussen et al, FEBS Lett 1996; 390(1):109
12). The sequence of HBP is publically available (for example as
NCBI accession no. NP_001691 REGION: 27 . . . 248) and is
reproduced below as SEQ ID NO.1
TABLE-US-00001 SEQ ID NO: 1
IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGV
STVVLGAYDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREAN
LTSSVTILPLPLQNATVEAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTP
EDQCRPNNVCTGVLTRRGGICNGDGGTPLVCEGLAHGVASFSLGPCGRGP
DFFTRVALFRDWIDGVLNNPGP
[0004] Urinary HBP levels in patients suspected of having a urinary
tract infection have not previously been investigated. The
inventors have shown for the first time that levels of HBP are
increased in individuals with urinary tract infection. According to
the invention there is thus provided a method of identifying
whether or not an individual has urinary tract infection, which
method comprises measuring HBP in the individual and thereby
determining whether or not the individual has urinary tract
infection.
[0005] The invention further provides: [0006] an agent for the
detection of HBP for use in determining whether or not an
individual has a urinary tract infection; [0007] a test kit for use
in a method for determining whether or not an individual has a
urinary tract infection, which test kit comprises an agent for the
detection of HBP in an individual; [0008] a method of treating an
individual for urinary tract infection comprising: [0009] (i)
determining whether or not an individual has a urinary tract
infection using a method of the invention; and [0010] (ii)
administering to an individual identified in (i) as at risk, a
therapeutically effective amount of at least one agent suitable for
the treatment of urinary tract infection.
DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows Urine levels of HBP, U-WBC and IL-6. Each dot
represents the concentration in an individual urine sample of HBP
(A and B), WBC (C and D) and IL-6 (E and F). The four patients
groups are described in the Methods section. Bars represent median
of values. The suggested cut-off value for HBP is marked at 32
ng/ml, for WBC at 2 WBC/.mu.L and for IL-6 10 pg/mL.
[0012] FIG. 2 shows Urinary levels of HBP and IL-6 correlated to
concentrations of bacteria in urine Each dot represents the
concentration in an individual urine sample of HBP (A) and IL-6
(B). Bars represent median of values.
[0013] FIG. 3 shows correlation between HBP and WBC in urine, and
between IL-6 and U-WBC in urine. Spearman's rank correlation
coefficient (rho)=0.79 indicate that there is a correlation between
levels of HBP and WBC in urine (A). Spearman's rank correlation
coefficient for IL-6 is 0.338. Each dot represents the
concentration in an individual urine sample of HBP, WBC and
IL-6.
[0014] FIG. 4 shows Receiver-operating characteristics curves (ROC)
of HBP and WBC in urine differentiating between UTI and no UTI.
Areas under the ROC curves (AUC) were 0.942 (95% confidence
interval, 0.889-0.996) for HBP, 0.808 (95% confidence interval,
0.675-0.941) for IL-6, 0.858 (95% confidence interval, 0.742-0.974)
for WBC.
DETAILED DESCRIPTION OF THE INVENTION
Diagnosis
[0015] The present invention relates to a method of identifying
whether or not a subject has a urinary tract infection. The
invention therefore relates to the diagnosis of urinary tract
infection.
[0016] The present inventors have for the first time investigated
the level of urinary HBP in patients suspected of having urinary
tract infection. The inventors have demonstrated that urinary HBP
levels are elevated in patients with urinary tract infection
relative to those who do not have urinary tract infection.
[0017] The individual under test is typically suspected of having a
urinary tract infection. The individual is typically a mammal. The
mammal is typically a human or a domestic mammal such as a horse, a
cow, a sheep, a dog or a cat. The individual is preferably a
human.
[0018] The individual under test may have one or more symptoms
associated urinary tract infections including for example fever,
typically greater than 37.5.degree. C., abdominal or back pain,
and/or a positive nitrite test.
[0019] The level of HBP is typically measured in vitro in a sample
obtained from an individual. The sample typically comprises a body
fluid of an individual. A fluid sample may be a sample of blood,
plasma, serum, urine, cerebrospinal fluid or joint fluid. The
sample is preferably a urine sample.
[0020] According to the present invention, an increased level or
concentration of HBP compared with the baseline level or
concentration indicates that the individual has a urinary tract
infection. The baseline level is typically the level of HBP in an
individual who does not have a urinary tract infection, or who is
suspected of having a urinary tract infection, but is subsequently
confirmed to not have a urinary tract infection. Thus the method
according to the present invention may include comparison of the
level of HBP in the sample to that of a baseline level.
[0021] In accordance with the present invention the concentration
of HBP is greater than 15 ng/ml, preferably greater than 25 ng/ml,
more preferably greater than 30 ng/ml, more preferably greater than
32 ng/ml, more preferably greater than 35 ng/ml, and most
preferably greater than 50 ng/ml in the sample. Such levels can be
used to indicate that an individual has a urinary tract
infection.
[0022] In an alternative aspect, the method may comprise assessing
the relative level or concentration of HBP in the sample compared
to a baseline level or concentration. Typically, a method wherein
the level or concentration of HBP in the sample is increased by at
least 3 fold or 4 fold relative to the baseline level or
concentration of HBP is indicative of urinary tract infection.
[0023] Other markers may also be included in the analysis such as
the white blood cell count (WBC), particularly the urine WBC,
and/or IL-6. HBP may also be used in combination with the nitrite
test.
Detection of HBP
[0024] The invention is typically carried out by measuring the
level of HBP in vitro in a sample obtained from the individual. The
sample typically comprises a body fluid of the individual. A fluid
sample may be a sample of blood, plasma, serum, urine,
cerebrospinal fluid or joint fluid. The sample is preferably a
urine sample. The sample may be processed prior to being assayed,
for example by centrifugation. The sample may also be typically
stored prior to assay, preferably below -70.degree. C.
[0025] Standard methods known in the art may be used to assay the
level of HBP. These methods typically involve using an agent for
the detection of HBP. The agent typically binds specifically to
HBP. The agent may be an antibody specific for HBP, an aptamer that
binds to HBP, a serine proteinase inhibitor such as aprotinin, for
example as described in Petersen et al, Eur J Biochem 1993; 271-9,
or a soluble fragment of an integrin, for example as described in
Cai and Wright, S. D. J Exp Med 1996; 184:213-23. By specific, it
will be understood that the agent or antibody binds to HBP with no
significant cross-reactivity to any other molecule, particularly
any other protein. For example, an agent or antibody specific for
HBP will show no significant cross-reactivity with human neutrophil
elastase. Cross-reactivity may be assessed by any suitable
method.
[0026] An antibody used in the method of the invention may either
be a whole antibody or a fragment thereof which is capable of
binding to HBP. The antibody may be monoclonal. Such a whole
antibody is typically an antibody which is produced by any suitable
method known in the art. For example, polyclonal antibodies may be
obtained by immunising a mammal, typically a rabbit or a mouse,
with HBP under suitable conditions and isolating antibody molecules
from, for example, the serum of said mammal. Monoclonal antibodies
may be obtained by hybridoma or recombinant methods.
[0027] Hybridoma methods involve immunising a mammal, typically a
rabbit, a rat or a mouse, with HBP under suitable conditions, then
harvesting the spleen cells of said mammal and fusing them with
myeloma cells. The mixture of fused cells is then diluted and
clones are grown from single parent cells. The antibodies secreted
by the different clones are then tested for their ability to bind
to HBP, and the most productive and stable clone is then grown in
culture medium to a high volume. The secreted antibody is collected
and purified.
[0028] Recombinant methods involve the cloning into phage or yeast
of different immunoglobulin gene segments to create libraries of
antibodies with slightly different amino acid sequences. Those
sequences which give rise to antibodies which bind to HBP may be
selected and the sequences cloned into, for example, a bacterial
cell line, for production.
[0029] Typically the antibody is a mammalian antibody, such as a
primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine,
equine or camel antibody. The antibody may be a camelid antibody or
shark antibody. The antibody may be a nanobody. The antibody can be
any class or isotype of antibody, for example IgM, but is
preferably IgG.
[0030] The fragment of whole antibody that can be used in the
method comprises an antigen binding site, e.g. Fab or F(ab)2
fragments or ScFV. The whole antibody or fragment may be associated
with other moieties, such as linkers which may be used to join
together 2 or more fragments or antibodies. Such linkers may be
chemical linkers or can be present in the form of a fusion protein
with the fragment or whole antibody. The linkers may thus be used
to join together whole antibodies or fragments which have the same
or different binding specificities, e.g. that can bind the same or
different polymorphisms. The antibody may be a bispecific antibody
which is able to bind to two different antigens, typically any two
of the polymorphisms mentioned herein. The antibody may be a
`diabody` formed by joining two variable domains back to back. In
the case where the antibodies used in the method are present in any
of the above forms which have different antigen binding sites of
different specificities then these different specificities are
typically to polymorphisms at different positions or on different
proteins. In one embodiment the antibody is a chimeric antibody
comprising sequence from different natural antibodies, for example
a humanised antibody.
[0031] Methods to assess HBP level typically involve contacting a
sample with an agent or antibody capable of binding specifically to
HBP. Such methods may include dipstick assays and Enzyme-linked
Immunosorbant Assay (ELISA). Typically dipsticks comprise one or
more antibodies or proteins that specifically bind HBP. If more
than one antibody is present, the antibodies preferably have
different non-overlapping determinants such that they may bind to
HBP simultaneously.
[0032] ELISA is a heterogeneous, solid phase assay that requires
the separation of reagents. ELISA is typically carried out using
the sandwich technique or the competitive technique. The sandwich
technique requires two antibodies. The first specifically binds HBP
and is bound to a solid support. The second antibody is bound to a
marker, typically an enzyme conjugate. A substrate for the enzyme
is used to quantify the HBP-antibody complex and hence the amount
of HBP in a sample. The antigen competitive inhibition assay also
typically requires an HBP-specific antibody bound to a support. An
HBP-enzyme conjugate is added to the sample (containing HBP) to be
assayed. Competitive inhibition between the HBP-enzyme conjugate
and unlabeled HBP allows quantification of the amount of HBP in a
sample. The solid supports for ELISA reactions preferably contain
wells.
[0033] The present invention may also employ antibodies to HBP in
direct sensing techniques including but not limited to those based
upon surface plasmon resonance, surface acoustic wave, quartz
crystal microbalance, microcalorimetry or electrochemical impedance
spectroscopy.
[0034] The present invention may also employ methods of measuring
HBP that do not comprise antibodies. High Performance Liquid
Chromatography (HPLC) separation and fluorescence detection is
preferably used as a method of determining the HBP level. HPLC
apparatus and methods as described previously may be used (Tsikas D
et al. J Chromatogr B Biomed Sci Appl 1998; 705:174-6) Separation
during HPLC is typically carried out on the basis of size or
charge. Prior to HPLC, endogenous amino acids and an internal
standard L-homoarginine are typically added to assay samples and
these are phase extracted on CBA cartridges (Varian, Harbor City,
Calif.). Amino acids within the samples are preferably derivatized
with o-phthalaldehyde (OPA). The accuracy and precision of the
assay is preferably determined within quality control samples for
all amino acids.
[0035] The invention further provides a diagnostic kit that
comprises means for measuring the HBP level in an individual and
thereby determining whether or not the individual has a urinary
tract infection. The kit typically contains one or more antibodies
that specifically bind HBP. For example, the kit may comprise a
monoclonal antibody, a polyclonal antibody, a single chain
antibody, a chimeric antibody, a CDR-grafted antibody or a
humanized antibody. The antibody may be an intact immunoglobulin
molecule or a fragment thereof such as a Fab, F(ab').sub.2 or Fv
fragment. If more than one antibody is present, the antibodies
preferably have different non-overlapping determinants such that
they may bind to HBP simultaneously.
[0036] The kit may additionally comprise means for the measurement
of other laboratory or clinical parameters. For example the kit may
comprise means for measuring the WBC count in an individual, and/or
the level or concentration of one or more of urinary nitrite, IL-6,
glucose, protein and plasma C-reactive protein.
[0037] The kit may additionally comprise one or more other reagents
or instruments which enable any of the embodiments of the method
mentioned above to be carried out. Such reagents or instruments
include one or more of the following: suitable buffer(s) (aqueous
solutions), means to isolate HBP from sample, means to obtain a
sample from the individual (such as a vessel or an instrument
comprising a needle) or a support comprising wells on which
quantitative reactions can be done. The kit may, optionally,
comprise instructions to enable the kit to be used in the method of
the invention or details regarding which individuals the method may
be carried out upon.
Therapy
[0038] The present invention also relates to the treatment of an
individual identified by a method of the invention as having a
urinary tract infection. Thus, a substance for use in the treatment
of a urinary tract infection may be used in the manufacture of a
medicament for use in the treatment of an individual identified by
a method of the invention as having a urinary tract infection. The
condition of an individual identified by a method of the invention
as having a urinary tract infection can therefore be improved by
administration of such a substance. A therapeutically effective
amount of a substance useful for the treatment of a urinary tract
infection may be given to an individual identified by a method of
the invention as in need thereof. Substances suitable for the
treatment of a urinary tract infection typically include one or
more antibiotics.
[0039] The following Example illustrates the invention:
Example
Methods
Study Population
[0040] This prospective study included 78 individuals (26 male and
52 female), who were admitted to the Department of Paediatric
Diseases, the Hospital of Helsingborg, Sweden, between March and
June 2009. The criteria for inclusion of patients in the study were
fever (temperature .gtoreq.37.5.degree. C.) or symptoms suggesting
urinary tract infection, abdominal or back pain in older
individuals and nonspecific signs such as irritability or
difficulties in feeding in younger children. Urinary tract
infection included both lower urinary tract infection and
pyelonephritis. Patients with neutropenia (neutrophils
.ltoreq.0.5.times.10.sup.9/L) or patients who had received
antibiotics in the previous 72 hours were excluded. The project
protocol was approved by the ethics committee of Lund University
Hospital and consent was obtained from all patients by their
parents. Patients were divided into the following groups: 1)
Patients with a final diagnosis of urinary tract infection, based
on bacterial culture, a urine sample with a single microorganism at
.gtoreq.10.sup.5 colony-forming-units (CFU)/ml and typical clinical
symptoms. 2) Patients with suspected urinary tract infection, based
on clinical characteristics or a urine sample with a single
microorganism .gtoreq.10.sup.3 CFU/ml, or a positive nitrite test.
The lower cut off value for bacteriuria is based on recommendations
from Swedish Institute for Infectious Disease Control. 3) Patients
with fever, but non-urinary tract infection 4) Patients without
fever, and no urinary tract infection.
Clinical and Laboratory Evaluations
[0041] Body temperature was recorded and CRP was analysed in
plasma. Urine was collected with midstream (75 samples) samples or
suprapubic aspiration (2 samples). In one patient the technique was
not registered. Analyses of urine samples were done with bacterial
cultures and dipstick tests, containing tests for nitrite, white
blood cells (WBC), albumin, red blood cells and glucose. Dipsticks
(Multisticks.RTM. 7, Siemens) were analysed on a Clinitek Status
(Bayer Health Care). Nitrite was presented as either positive or
negative, and WBC as semi-quantitative values in a graded scale:
0<15 leukocytes/.mu.L urine, 1.gtoreq.15 leukocytes/.mu.L urine,
2.gtoreq.70 leukocytes/.mu.L urine, 3.gtoreq.125 leukocytes/.mu.L
urine, 4.gtoreq.500 leukocytes/.mu.L urine. For determination of
HBP levels, urine was centrifuged within one hour of the sampling,
and aliquots of the supernatants were stored at -70.degree. C.
until analysis. The concentration of HBP was determined by
enzyme-linked immunosorbent assay (ELISA). Microtiter plates (NUNC)
were coated with a mouse monoclonal antibody directed against HBP
in coating buffer (0.05M NaHCO.sub.3, pH 9.6). Plates were washed
with phosphate-buffered saline and blocked with bovine serum
albumin (Sigma). Urine samples were diluted 1/40 in sample buffer
(1M NaCl), added to the wells in duplicate, and incubated for 30
min at 37.degree. C. Each plate also contained calibration samples
of known concentration of recombinant HBP. After washing, plates
were incubated with a polyclonal rabbit antiserum towards HBP
diluted 1/7000. Bound antibodies were detected by incubation with
peroxidase-conjugated antibody against rabbit IgG, Bio-Rad
(1/3000). The level of HBP in each patient sample was determined by
calculating the mean optical densities of duplicates which were
correlated to the results from the standard curve. Levels of IL-6
in urine were analysed with a sandwich-ELISA, (ELISA MAX.TM. Deluxe
Sets), according to the manufacture's description. Urine samples
were diluted 1/2 in incubation buffer, and analysed in
duplicates.
Statistical Analysis
[0042] Comparisons between groups were made by the non-parametric
Kruskal-Wallis test and Mann-Whitney-U-test for continuous
variables with skewed distributions. Correlations were made by
Spearman's non-parametric test. Areas under receiver-operator
characteristic curves were used to illustrate the power of HBP,
IL-6, U-WBC and nitrite. Sensitivity, specificity, positive and
negative predictive value were calculated for HBP, IL-6 and U-WBC.
The level of statistical significance was defined as two-tailed
p-value<0.05. The SPSS 17.0 software system (SPSS) was used for
statistical analyzes.
Results
Characteristics of the Patients and Laboratory Signs
[0043] Seventy eight individuals were enrolled in the study. The
age of the patients ranged from 1 month to 18 years (mean 5.96
years) and 75% were older than 1 year. Ten patients were diagnosed
with urinary tract infection (2 with lower urinary tract infection
and 8 with pyelonephritis), and all had Escherichia coli in the
urine culture (group 1). Five patients had a suspected urinary
tract infection based on characteristic symptoms or laboratory
results indicating urinary tract infection (group 2). Three of
these 5 patients had only a moderate growth of Escherichia coli or
beta-hemolytic Streptococci group B in urine
(.gtoreq.10.sup.3-.ltoreq.10.sup.5 CFU/ml), one patient had typical
clinical symptoms of cystitis but were not cultured and had a
negative dipstick test, and one patient had characteristic signs of
pyelonephritis with a negative urine culture and a dipstick test
that was positive for nitrite. Thirty patients had fever but no
laboratory or clinical signs of urinary tract infection (group 3).
Their urine cultures were negative or had mixed strains of bacteria
(indicative of sample contamination). Thirty-three patients had no
fever and no laboratory signs of urinary tract infection (group 4).
Their urine cultures were negative. Some of these had diagnoses
(known before entering the study) like enuresis, Alport syndrome,
Henoch-Schonlein-syndrome and nephrotic syndrome. Statistical
Analysis of Results from Plasma and Urine There were no significant
differences in median age, gender, temperature, CRP in plasma, HBP
in urine, IL-6 in urine, WBC or nitrite in urine between group 1
(urinary tract infection) and 2 (suspected urinary tract
infection). Plasma CRP and urinary glucose did not result in any
significant differences between any groups. Urinary albumin had
significant differences between group 1 and 3 (fever, non-urinary
tract infection), 1 and 4 (no fever, non infection), 2 and 3, but
not between the others. Urinary red blood cells had significant
differences between group 1 and 3, 1 and 4 but not between the
others. There were significant differences in median levels of HBP,
IL-6 and WBC in urine between group 1 and 3. Also comparison of
group 1 and 4 had significantly different median levels of HBP,
IL-6 and WBC in urine. There were significant differences in the
urine levels of HBP, IL-6 and WBC between group 1 combined with
group 2 as compared to group 3 combined with group 4. (FIG. 1 A-F).
An interesting implication was that urine with high concentrations
of bacteria (.gtoreq.10.sup.5 CFU/ml) seemed to have higher levels
of HBP and WBC compared to urine with less or no bacteria (FIG. 2
A-B). Spearman's test with a rank correlation coefficient
(rho)=0.79 indicated a relatively strong correlation between levels
of HBP and WBC in urine (FIG. 3). IL-6 did not have this strong
correlation with WBC in urine, (rho=0.38).
[0044] For calculation of sensitivity, specificity, positive
predictive value and negative predictive value group 1 and 2 was
combined, referred to urinary tract infection-group and compared to
the combined groups 3 and 4, referred to non-urinary tract
infection. Using a cut-off level of HBP of .gtoreq.32 ng/ml, the
sensitivity and specificity in diagnosing urinary tract infection
were 93.3% and 90.3% respectively. HBP in urine had the highest
sensitivity compared to WBC, nitrite and IL-6. HBP in urine also
had the highest specificity compared to WBC and IL-6 (Table 2).
Nitrite had the best specificity compared to the other markers.
Based on the 19.2% prevalence of urinary tract infection in this
study the positive and negative predictive values of HBP for
prediction of urinary tract infection were 70.0% and 98.3%,
respectively (Table 2). ROC curves demonstrated that HBP in urine
was the best predictor of urinary tract infection with an area
under curve (AUC) value of 0.94 compared to U-WBC and IL-6 (FIG.
4).
Discussion
[0045] Urinary tract infection can have serious consequences and
antibiotic resistance is a growing issue. Therefore, tests with
higher sensitivity and specificity are needed. Several previous
studies have investigated biomarkers in urine for the detection of
urinary tract infection, like IL-6, lactoferrin, IL-8, TNF-.alpha.,
elastase and Clara cell protein. HBP in plasma has been studied as
a biomarker for sepsis in adults, and data suggested that a normal
plasma level of HBP in febrile patients with suspected infection
could with high probability (negative predictive value 94.5%) rule
out the risk of developing severe sepsis. In the present study,
concentrations of HBP in urine was compared with urinary IL-6,
nitrite, WBC, red blood cells, albumin, glucose and plasma CRP. HBP
in urine had a higher AUC value (0.94) than IL-6, nitrite and WBC,
and was thus the best predictor for urinary tract infection. HBP in
urine had a high negative predictive value (98.3%), and could thus
rule out disease with a high probability. The nitrite test has its
limitations with a low sensitivity. Some bacterial species can not
transform nitrate to nitrite, for example Staphylococcus
saprophyticus and Enterococcus. Other reasons for false negative
nitrite are short incubation time of urine in the bladder and
intake of high doses of vitamin C. Outliers in the study exist. In
group 2 (suspected urinary tract infection), one patient with
symptoms of pyelonephritis and beta-hemolytic Streptococci group-B
in the urine culture (10.sup.4-10.sup.5 CFU/ml) had a low level of
HBP (6.3 ng/ml). This patient was also negative in the rapid test
for urinary nitrite and WBC. Urine HBP levels were higher in
patients with urinary tract infection than in patients with other
kinds of infections, which indicates that HBP release in urine is
dependent on the activation of white blood cells by for instance
bacterial infection in urine.
TABLE-US-00002 TABLE 1 Patient Characteristics. Clinical and
laboratory data. Median, range and P-values for age, gender, CRP,
HBP, temperature, U-WBC and nitrite. Group 1 Group 2 Group 3 Group
4 P-value P-value P-value (n = 10) (n = 5) (n = 30) (n = 33) x xx
xxx Age 4.5 (0-17) 7.36 (0-16) 2.0 (0-17) 8.1 (0-18) 0.322 0.225
0.299 (years) Gender 9/1 4/1 23/7 16/17 0.604 0.367 0.021
(female/male) HBP 72.1 (37.0-632.2) 108.3 (6.4-230.9) 5.3
(3.3-121.2) 4.3 (3.2-76.5) 1.00 0.000 0.000 (ng/ml) U-WBC.sup.1 3
(2-4) 1 (0-3) 0 (0-3) 0 (0-2) 0.077 0.000 0.000 (WBC/.mu.L) Nitrite
1 (0-1) 0 (0-1) 0 (0-0) 0 (0-0) 0.077 0.000 0.000 (pos/neg).sup.2
Temperature 37.9 (36.7-40.0) 38.5 (37.3-41.1) 38.2 (37.0-41.1) 37.0
(36.9-39.4) 0.297 0.171 0.000 (.degree. C.) CRP 146 (0-201) 165
(10-184) 56 (0-190) 16 (0-169) 0.322 0.152 0.013 (ng/ml) NOTE:
P-values analyzed with the Mann-Whitney test. x = Group 1 vs 2, xx
= Group 1 vs 3, xxx = Group 1 vs 4 .sup.1Semi-quantitative scale: 0
< 15 leukocytes/.mu.L, 1 .gtoreq. 15 leukocytes/.mu.L, 2
.gtoreq. 70 leukocytes/.mu.L, 3 .gtoreq. 125 leukocytes/.mu.L, 4
.gtoreq. 500 leukocytes/.mu.L .sup.2Positive nitrite was defined as
1 and negative nitrite was defined as 0.
TABLE-US-00003 TABLE 2 Sensitivity, Specificity, PPV and NPV of
HBP, U-WBC and nitrite for predicition of UTI. (Cutoff Values of 32
ng/mL for HBP, 10 pg/mL for IL-6, 2 for U-WBC, 100 ng/ml for CRP,
0.5 for nitrite, 38.degree. C. for temperature). Sensitivity, %
Specificity, % PPV, % NPV, % HBP 93.3 90.3 70.0 98.3 IL-6 73.3 84.1
52.4 93.0 U-WBC 80.0 84.8 57.1 94.3 Nitrite 53.3 100 100 89.4 CRP
64.3 72.7 50 82.8 Temperature 53.3 63.3 23.7 84.4 PPV, positive
predictive value, NPV, negative predictive value.
Sequence CWU 1
1
11222PRThomo sapiens 1Ile Val Gly Gly Arg Lys Ala Arg Pro Arg Gln
Phe Pro Phe Leu Ala 1 5 10 15 Ser Ile Gln Asn Gln Gly Arg His Phe
Cys Gly Gly Ala Leu Ile His 20 25 30 Ala Arg Phe Val Met Thr Ala
Ala Ser Cys Phe Gln Ser Gln Asn Pro 35 40 45 Gly Val Ser Thr Val
Val Leu Gly Ala Tyr Asp Leu Arg Arg Arg Glu 50 55 60 Arg Gln Ser
Arg Gln Thr Phe Ser Ile Ser Ser Met Ser Glu Asn Gly 65 70 75 80 Tyr
Asp Pro Gln Gln Asn Leu Asn Asp Leu Met Leu Leu Gln Leu Asp 85 90
95 Arg Glu Ala Asn Leu Thr Ser Ser Val Thr Ile Leu Pro Leu Pro Leu
100 105 110 Gln Asn Ala Thr Val Glu Ala Gly Thr Arg Cys Gln Val Ala
Gly Trp 115 120 125 Gly Ser Gln Arg Ser Gly Gly Arg Leu Ser Arg Phe
Pro Arg Phe Val 130 135 140 Asn Val Thr Val Thr Pro Glu Asp Gln Cys
Arg Pro Asn Asn Val Cys 145 150 155 160 Thr Gly Val Leu Thr Arg Arg
Gly Gly Ile Cys Asn Gly Asp Gly Gly 165 170 175 Thr Pro Leu Val Cys
Glu Gly Leu Ala His Gly Val Ala Ser Phe Ser 180 185 190 Leu Gly Pro
Cys Gly Arg Gly Pro Asp Phe Phe Thr Arg Val Ala Leu 195 200 205 Phe
Arg Asp Trp Ile Asp Gly Val Leu Asn Asn Pro Gly Pro 210 215 220
* * * * *