Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT03252028 |
| Other study ID # |
LINPEAS |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
August 13, 2017 |
| Last updated |
August 18, 2017 |
| Start date |
December 2, 2015 |
| Est. completion date |
December 1, 2020 |
Study information
| Verified date |
August 2017 |
| Source |
University Hospital, Linkoeping |
| Contact |
Fariba Nayeri, MD |
| Phone |
+46702080804 |
| Email |
fariba.nayeri[@]regionostergotland.se |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Background: Due to anatomical restrictions, the inflammatory response to intra-cerebral
bacterial infections exposes swollen brain tissues to pressure and ischemia, resulting in
life-threatening damage. However, diagnosing meningitis in patients after neurosurgery is
complicated, due to brain tissue damage and changes in cerebrospinal fluid (CSF) caused by
surgery. Hepatocyte growth factor (HGF) is a local, acute-phase protein. Previous studies on
community-acquired septic meningitis reported high levels of intrathecal-produced HGF.
Aim: The aim of present study is to evaluate a new platform for qualitative determination of
HGF in body fluids and revealing the site of injury.
Method: Based on a reverse-methachromacy method, strips are prepared. The surface on the
strip changes colour to blue upon contact with HGF.
Plan: CSF, urine and sputum of patients that develop fever post neurosurgery are analysed
with the test and the results compared with conventional diagnostic methods.
Clinical value: A rapid, equipment-free test gives the opportunity to identify the infectious
focus in the infected organ long before culture results are available.
Description:
Hepatocyte growth factor (HGF) is local acute phase protein with regenerative properties that
become biologic inactive during chronic inflammation and loses the binding affinity to
glycosaminoglycan in the extracellular ma-trix. HGF is excreted into the gastrointestinal
tract and is not detected in normal urine. Nosocomial meningitis can occur when brain
surgical procedures are complicated by infection. Due to underlying CNS disease processes and
CNS devices in situ, the principal agents of nosocomial bacterial meningitis differ from the
agents of community-acquired meningitis. For example, in nosocomial infections, slow growing,
opportunistic microorganisms predominate. The CSF leukocyte profile is affected by
intracerebral haemorrhage, and CSF lactate might be elevated, due to ischemia. Moreover,
altered consciousness can make it difficult to establish a diagnosis in patients on
ventilators that develop fever after neurosurgical operations. In other words, it is often
difficult to determine whether the injured brain has been invaded by environmental bacterial
flora.
Due to the challenges in establishing a diagnosis, and the lack of gold standards, physicians
are motivated to treat suspected infections in serious dis-eases with broad-spectrum
antibiotics. The emerging problems of multiple-resistance bacteria, high costs, and
complications related to new antibiotics have called for diagnostic tests that can minimize
antibiotic consumption.
Currently, the diagnosis of bacterial meningitis remains based on standard methods of direct
microscopy, differential analyses of white blood cells, lactate, and protein, and cultures of
blood and CSF . However, post-neurosurgical infections are difficult to distinguish from the
effects of neurosurgical procedures. Moreover, due to prophylaxis treatments, the cultures
are negative in a large group of patients, and the presence of skin flora, like Coagulase
negative Staphylococcus or Propionbacterium acnes, may indicate either infection or
contamination. Survival from this life-threatening condition depends on a rapid diagnosis and
prompt empirical antibiotic therapy designed to cover the likely pathogens.
Where is the focus of infection? Is it a bacterial (septic) meningitis? Is the broad-spectrum
antibiotic administration indicated? The background of the project: An invasion of bacteria
into the central nervous system (CNS) is followed by a rapidly evolving inflammatory process
that affects the arachnoid space, the pia mater, and the cerebrospinal fluid (CSF). This
condition leads to clinical symptoms of headache, fever, and meningism. The inflammatory
response is caused by the release of various pro-inflammatory cytokines from meningeal cells
into the subarachnoid space. As a result, neutrophils move into the subarachnoid space and
cause pleocytosis in the CSF. The consequences include the breakdown of the blood-brain
barrier, cerebral oedema, reduced cerebral blood flow, focal areas of hypo perfusion,
vascular thrombosis, ischemia, enhanced glucose metabolism via the anaerobic glycolytic
pathway, and enhanced lactate accumulation in the brain and CSF [1]. Survival from this
life-threatening condition depends on a rapid diagnosis and prompt empirical antibiotic
therapy designed to cover the likely pathogens.
Other causes of febrile meningitis include acute viral meningitis and non-pyogenic
meningitis, where the clinical picture is typically sub-acute or chronic. The diagnostic
procedures consist of a lumbar puncture to analyse the CSF for cells and bacteria,
microbiological cultures of blood and CSF, serological tests involving PCR and
antigen-detection, and radiographic techniques [2]. In community-acquired meningitis, a
combination of discriminating values from the CSF analysis can differentiate acute bacterial
meningitis from other, non-ambulatory causes with quite high sensitivity [3].
Nosocomial meningitis can occur when brain surgical procedures are complicated by infection
[2]. Due to underlying CNS disease processes and CNS devices in situ, the principal agents of
nosocomial bacterial meningitis differ from the agents of community-acquired meningitis. The
CSF leukocyte profile is affected by intra-cerebral haemorrhage, and CSF lactate might be
elevated, due to ischemia. Moreover, altered consciousness can make it difficult to establish
a diagnosis in patients on ventilators that develop fever after neurosurgical operations.
Hepatocyte growth factor (HGF) is produced by mesenchymal cells during organ injury. It is
produced as a single-chain precursor protein, and it is activated at the site of injury by
proteolysis cleavage, resulting in a double-chained active form of HGF [4]. Active HGF
stimulates cell division [4] and cell motility, and it promotes normal morphogenic structure
[5] in epithelial cells adjacent to injured areas. Thus, HGF induces regeneration and re-pair
of damaged tissue [6]. High levels of systemic HGF have been detected during injuries caused
by infection [7]. In bacterial meningitis, pneumonia and acute bacterial gastroenteritis,
there is local production of HGF at the site of infection [7-9]. HGF produced locally during
bacterial infection is biologically active [10]. Application of biologically active HGF
promoted healing of chronic leg ulcers in a pilot study [11]. Effective antibiotic therapy
reduces systemic HGF levels during infection [12-13]. HGF might be regarded as a local acute
phase protein with healing properties [14].
The quality of endogenous HGF binding to receptors can be assessed with surface plasmon
resonance (SPR), an optical technique appropriate for clinical studies that can determine the
affinity of a protein for several ligands [15]. SPR-based assessment of HGF binding affinity
for its receptors, c-Met and heparan sulphate proteoglycan (HSPG), could rapidly and
sensitively distinguish HGF variants with different biological activities [16-17].
We have previously studied the concentration of HGF in CSF for patients with community-
acquired meningitis [7]. We have also shown that the presence of biologically active HGF at
the site of injury indicates an acute local inflammation [18]. During a recent study we have
assessed whether HGF concentrations and HGF binding affinity for its receptors might serve as
markers to distinguish between meningitis associated with neurosurgery and other causes of
infection. We determined the concentration and binding affinity of HGF in patients with
either community-acquired meningitis or neurosurgery-associated meningitis, and compared the
results to either patients with Alzheimer's disease or controls with normal CSF. We have
shown that determination of HGF in CSF might be used as an indicator, complementary to
clinical status and routine laboratory findings, for diagnosing bacterial invasion into the
CSF at an early stage of disease [18].
The problem:
Case report: 76 years old woman with high blood pressure was found by her neighbours,
unconscious in her garden 28th October 2009. The CT scan revealed subarachnoid haemorrhage
and neuro-angiography revealed 3 aneurysms. She was admitted to the Department of
neurosurgery and operated 29th October with ventricular drainage and endovascular occlusion
of the aneurysms. She was extubated post-surgery however she suffered from left arm and leg
paralysis and tiredness. Since 30th October she had low grade of fever and slightly increased
CRP and procalcitonin. Cerebrospinal fluid showed 170.000 red and 630 white blood cells and
lactate 5.8. The chest X-ray showed a suspect infiltration. The blood and CSF cultures were
secured. The cultures taken from 5th November revealed significant growth of Enterobacter
cloacae and Staphylococcus aurous in urine and growth of Staphylococcus aurous in the
nasopharynx. She received no antibiotic therapy and the parameters decreased spontaneously
but low grade of fever continued. The CSF blood cells increased to 23000 red and 106 white
blood cells (11th November), and CSF lactate was 4.5 (lumbar drainage). Although CRP was in
the normal range she received meropenen 2g x 3 under suspicion bacterial meningitis. First on
19th November the CSF cultures taken on 11th November revealed growth of Propionibacterium
acnes. The antibiotic regime continued until 23th November. She suffered from disorientation,
fatigue, and had hydrocephalus. She underwent shunt operation 23th November. She died 2nd
December 2009. High concentration of HGF and elevated binding affinity to HSPG was found in
CSF analysis already 2nd November.
Due to the challenges in establishing a diagnosis, and the lack of gold standards, physicians
are motivated to treat suspected infections individually because there is no diagnostic
method that can identify the infections focus in time. PCR for bacterial panel and virus
detection in CSF is sensitive method. However it might detect the colonized bacteria as well.
The presence of bacteria is not the same as infection. Therefore in cases of bacterial
meningitis especially post brain surgery and the foreign bodies that are inserted in the
brain we cannot rely on positive or negative cultures totally. A complementary method (for
the standard available methods at laboratory) with high negative predictive value increases
the specificity of tests and decreases the costs, time and complications such as hospital
acquired infections (HAI) profoundly.
The Method, Reverse Methachromacy:
The N-terminus of HGF (Hairpin Loop) has been identified as the binding site to both c-Met
and HSPG. There is a high correlation between binding of HGF to HSPG and to dextran sulphate
in SPR system (fig 1). Metochromasia is a characteristic colour change that certain aniline
dyes exhibit when bound to chromo trope substances [19].This phenomenon has been widely used
in the study of tissue sections. Methylene blue (O-toluidine) is considered an excellent
metachromatic dye. Upon binding to high molecular weight polysaccharides, such as dextran
sulphate, the colour of the indicator solution changes from blue to red [19].
Combinations of dextran sulphate and O-toluidine in different proportions produce purple-red
coloured solutions of varying intensity. Coated customized filter papers make up surfaces
that are placed on strips. When in con-tact with biological solutions, the sample HGF
competitively replaces O-toluidine for binding to dextran sulphate and the colour of the
surface returns to blue (Fig 2). This platform detects the presence of HSPG-binding proteins
such as HGF in body fluids including expectorant, urine, ulcer secretions, cerebrospinal
fluid, and joint effusion during infection. High amounts of protein or high pH do not cause
non-specific reactions with this device.
The study plan:
Study leader: Amir Ramezani
The Procedure:
CSF specimens, urine and sputum (n=500) are collected from patients (1-99 years old) that
have undergone brain tumour surgery, intracerebral haemorrhage, shunt dysfunction or skull
fracture. The specimens are kept frozen in −20° C until analyses were performed. There are no
exclusion's criteria.
Controls Alzheimer's disease (n=20)
- CSF samples were collected and kept frozen from patients (N=20) that were diagnosed with
Alzheimer's disease at the Memory Clinic, Skåne University Hospital in Malmö. The
revised DSM-III and NINDS-ADRDA criteria were used for diagnosis of Alzheimer's dementia
(this study group has been included in previous study).
- Normal CSF
- This group consists of patients who have undergone lumbar puncture to rule out
meningitis, and they had normal CSF.
- Analysis of ligand-binding affinity with surface plasmon resonance
- The biological activity of HGF is analysed with SPR. We measure the binding affinity to
HSPG (Sigma-Aldrich, St. Louis, MO, USA) and to a c-Met recombinant chimera (R&D
Systems), as previously described.
- Measurements of HGF concentrations in samples A specific enzyme-linked immunosorbent
assay (ELISA) kit (Quantikine Human HGF immunoassay, minimum detectable limit: 0.04
ng/mL; R&D Systems, Minneapolis, MN, USA) was used to determine HGF concentrations in
CSF, sputum and urine according to the manufacturer`s instructions. All measurements
were performed with an ELISA reader (Expert96; AsysHitech GmbH, Eugendorf, Austria) at
450 nm, which was calibrated with the recombinant human HGF reference samples and the
standards provided in the kit.
- Reverse methachromacy The study strip test is performed on all specimens.
- Routine laboratory assessments
- CSF is analysed to determine parameter values at the Departments of Clinical Chemistry,
University Hospitals, in Linköping. All cultures, PCR assays, antigen detection assays,
and serological assessments are performed at the Department of Microbiology, University
Hospital, Linköping, Sweden.
- CSF-cells: Performed by manual phase contrast microscopy (Zeiss) using Jessen Chamber
for counting the number of erythrocytes and leukocytes (polymorphonuclear neutrophils
and monocytes).
- CSF lactate: Performed using benchtop blood gas analyzer ABL 800 (Radiometer Medical ApS
Denmark).
- Antigen detection: Antigen (Streptococcus pneumonia, Haemophilus influenza, Neisseria
meningitides group A, group B/Escherichia coli K1, group C and group Y/135,
Streptococcus agalactiae) is detected by latex agglutination method Pastorex Meningitis™
(Bio-Rad, France).
- CSF culture: Performed in aerobe and anaerobe flasks as well as in Hematin plates.
- Virus detection PCR: Herpes virus type 1 and 2 (HSV1, HSV 2) and HZV DNA were detected
by PCR. Human enterovirus (HEV) was analysed by automated instrument (Cepheid
GeneXpert).
- Statistical analysis
- Because the HGF concentrations and binding affinity data are not normally distributed,
non parametric tests Kruskal-Wallis test followed by the Mann-Whitney U test or the
Wilcoxon matched pairs test are appropriate for analysis. The analysis of test
performance shall be performed manually.
Clinical Evaluation of the Platform in a Dextran Sulphate-Containing Gel Using reverse
methachromacy approach we have studied a total of 656 faeces samples with The sensitivity,
specificity, positive predictive value, and negative predictive value as well as the accuracy
were calculated in the following groups: verified infectious gastroenteritis (n=207) and
other causes of diarrhoea, including IBD (n=268). The test could distinguish acute infectious
gastroenteritis with a sensitivity of 96.6% and specificity 92.4%, and a positive predictive
value of 90.9% and negative predictive value of 97.2%. The accuracy of test was 94.3%. No
significant correlation between the test results and faeces calprotectin were found
(R2=0·056). No significant correlation was observed between the results of the index test and
the presence of blood in the faeces (R2=0·08) [20].
Clinical evaluation of HGF as a marker to distinguish septic meningitis from other causes of
pleocytosis [18] The community-acquired septic meningitis showed significantly higher HGF
concentration (p=0.0133), as well as HGF binding affinity to the c-Met and HSPG receptors
(p=0.0007 and p=0.0009, respectively) compared to nosocomial meningitis. CSF samples from
patients with septic meningitis (including both community-acquired and nosocomial) was
significantly higher in HGF concentrations (p=0.0014), HGF binding to HSPG (p<0.0001), and
HGF binding to c-Met (p<0.0001) compared to samples from patients with aseptic (viral and sub
acute) meningitis. CSF samples from patients with septic meningitis was higher from samples
from the control group (patients with normal CSF) and from patients with Alzheimer's disease
in HGF concentration (p<0.0001, p=0.0010, respectively), HGF binding to HSPG (p<0.0001 and
p=0.9, respectively), and HGF binding to c-Met (both p<0.0001).
Compared to samples from patients that had undergone neurosurgery and had other infectious
diseases, CSF samples from patients with nosocomial meningitis had significantly higher HGF
concentrations (p<0.0001) and HGF binding affinity to c-Met (p<0.0001) and HSPG (p=0.043)
receptors (Figure 3-4).
Figure Legends Fig. 1. Sequence of part of the HGF α-chain. The enlarged codons code for
amino acids important for binding to HSPG and the c-Met receptor.
Fig. 2. The theoretical support for the second approach for a device to de-tect the presence
of HGF in body fluids during an infection. (1) Glucosaminoglycan (GAG) polymer chain with
negative charges. (2) Positive TBO molecules attach to the polymer chain then are
concentrated and form stacks. The colour changes from blue to red. (3) A sample with HGF is
added, which binds to GAG. (4) TBO molecule stacks are destroyed and become freely dissolved
TBO molecules. The colour changes from red to blue.
Figure 3: Flow chart of the selection of CSF specimens from patient and control groups. Extra
samples are the samples taken during stay on ward from patients with nosocomial meningitis.
Figure 4: Properties of HGF derived from different CSF samples were analysed by using surface
plasmon and ELISA techniques. (A) Binding to c-Met receptors; (B) Binding to HSPG receptors;
(C) HGF concentrations (median). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
The rapid evolution and spread of antimicrobial-resistant bacteria in parallel with
insufficient development of new active drugs seriously affect future anti-infective therapy
of bacterial infections, especially those due to Gram-negative rods. Experts in the field
estimate that by the next decade the world will have witnessed the wide dissemination of
untreatable (or next-to untreatable) infections, both within and beyond hospitals. To avoid
or at least attempt to retard this crisis, many researchers have dedicated their efforts to
elucidate factors favouring the emergence and global spread of antimicrobial resistant
bacteria. An effective strategy to limit the emergence of multiple resistant strains might
include directing the antibiotic therapy by identifying cases that should or should not be
treated with antibiotics, avoiding wide-spectrum antibiotic administration, and promoting
more specific treatments.
What is needed is a simple, accurate, cost-effective and feasible test that answers to
crucial questions.
In order to assess the clinical relevance and the performance of such a test there is not a
better environment than the Swedish healthcare that provides the relevant and modern
assessment of diseases and evidence-based therapy for nearly all patients. Such an
environment is an opportunity to perform the patient inclusion and evaluation of the study
test in comparison to the most reliable standard tools such as cultures, blood tests and
X-rays. These resources are not available automatically in any other centre with such
reliable, non-commercial and impartial properties.
dnr 2015-429-32
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