Clinical Trial Details
— Status: Active, not recruiting
Administrative data
| NCT number |
NCT02153034 |
| Other study ID # |
MR/J01477X/1 |
| Secondary ID |
|
| Status |
Active, not recruiting |
| Phase |
N/A
|
| First received |
May 28, 2014 |
| Last updated |
March 22, 2017 |
| Start date |
May 2013 |
| Est. completion date |
January 2018 |
Study information
| Verified date |
March 2017 |
| Source |
Kwame Nkrumah University of Science and Technology |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans
(Mu) in rural parts of West Africa. It causes large skin ulcers mainly in children aged 5 to
15 years. Access to treatment is limited and many cases present late. There have been major
advances in understanding the mechanism of disease together with improved diagnosis and
management. The aim of the proposed studies is to identify markers predictive of a rapid
response to antibiotic treatment and to investigate the pathogenesis of paradoxical
reactions and oedematous lesions in Mu disease.
Infection with Mu results in a nodule under the skin which enlarges and breaks down to form
an ulcer. This is because Mu produces a toxin that spreads outwards and damages subcutaneous
tissue. In recent years it has been found that antibiotic treatment for 8 weeks with daily
tablets and intramuscular injections heals ulcers. This is unpleasant and it would be better
if the treatment could be shortened. Our previous studies suggest this may be possible.
Therefore a wide range of tests will be investigated in order to identify markers for people
in whom the infection is at an early stage with low numbers of Mu bacteria and low levels of
toxin in the skin. During antibiotic treatment the rate of healing will be measured to find
out which markers are the most reliable.
In some patients new areas of inflammation develop despite treatment and this is called a
paradoxical reaction. The immune response to Mu will be investigated serially during
antibiotic treatment to investigate the cause of paradoxical reactions.
About 15% of patients have oedematous disease, the most severe form of Buruli ulcer. We will
study the amount of Mu toxin produced by the strain of Mu cultured from patients with this
form of the disease.
Hypothesis
- Buruli ulcer patients that heal rapidly/slowly or develop paradoxical reactions with
treatment will have associated predictive viability or serum biomarkers.
- Buruli ulcer patients with oedematous disease are associated with larger amounts of
mycolactone and viable organisms
Description:
Procedures
Infected Participants
After collection of demographic data using standard World Health Organisation forms (BU01)
together with a careful history to establish when early lesions (nodules, plaques and
ulcers) were first observed, the type and dimensions of lesions will be documented together
with digital photographs and tracings onto acetate sheets. For oedematous lesions only
digital photographs will be obtained. Patients will be reviewed at 2 weekly intervals during
standard antibiotic treatment (rifampicin 10mg/kg and streptomycin 15mg/kg) with further
recordings of clinical data as is done for all routine patients. These measurements will
enable calculation of rate of healing and healing time in relation to lesion size and type.
Patients will be monitored for paradoxical reactions that occur after the start of treatment
in about 8% of patients. These procedures are routinely provided as part of routine care of
Buruli ulcer patients.
Samples
The additional samples required from patients for the study are an extra volume of blood
(7ml) and 3 swabs taken on occasions (at baseline, week 4, 8, 12 and 16) only when the
patient has a lesion at these time points. Swabs/fine needle aspirates are required to
determine if organisms detected by polymerase chain reaction (PCR) are still viable, one for
M. ulcerans culture, one for measurement of mycolactone concentration, and one for combined
16S ribosomal ribonucleic acid (rRNA) reverse transcriptase / insertion sequence IS2404
Real-Time qPCR assay (1).
For patients that develop paradoxical lesions during therapy an additional blood sample and
3 swabs/fine needle aspirates will be obtained at the time of the reaction for viability
markers (M. ulcerans culture, combined 16S rRNA reverse transcriptase / IS2404 Real-Time
quantitative polymerase chain reaction (qPCR) assay and mycolactone detection).
For oedematous lesions, one swab/fine needle aspirates will be obtained for mycolactone
detection and quantification from lesions that are ulcerated. A 3 mm punch biopsy will be
performed under local anaesthetic on non-ulcerated lesions for the same purpose only when
the patient is 15 years old or above.
Established practice for routine diagnosis of M. ulcerans infection is to take fine needle
aspirates from non-ulcerated lesions for acid-fast bacillus (AFB) detection, culture and PCR
for IS2404. This specimen is not large enough for mycolactone quantification, an important
part of the investigation into the pathogenesis of oedematous disease which often presents
without ulceration. Swabs are used in the diagnosis of ulcers.
Controls For the household contacts of patients in endemic villages and healthy controls in
non-endemic villages a 7ml blood sample will be obtained to investigate protein biomarkers
by Luminex assay, T cell subsets by flow cytometry and immune responses using Enzyme Linked
Immunosorbent Assay(ELISA) in a whole blood assay for comparative studies. No tissue
biopsies will be taken.
Definition of paradoxical reaction Paradoxical reaction will be defined as an increase in
inflammatory changes with increase in lesion size of greater than 100%, after initial
improvement and decrease in size; and/or the appearance of new lesions following or during
antimycobacterial treatment.
Investigations Detection of rapid responders: Biomarkers, tissue mycolactone concentration
and immune response will be measured at baseline. The rate of healing will be estimated by
documenting the initial size of the lesion and the time to complete healing as previously
(2).
Detection of Mu: Swabs homogenates will be inoculated onto Lowenstein Jensen slopes for
culture and microscopy will be carried out on Ziehl-Neelsen stained smears for AFB. PCR for
IS2404 will be carried out as described previously (3).
Biomarkers:
Serum samples will be subjected to multi-analyte profiling using Luminex multianalyte
profiling to identify markers of oedematous disease, paradoxical reactions and rapid
responders.
We will also use mass spectrometry-based proteomics to generate additional serum/tissue
biomarkers taking a three-stage approach. First we will use sample fractionation and mass
spectrometry (matrix-assisted laser desorption/ionisation/time of flight/mass spectrometry)
to determine the degree of variability of proteomic composition in samples from subjects to
be treated with antibiotics. This first screening will allow the identification and removal
of subjects showing a partial response and subsequent analysis of those showing the most
disparate responses. Stage 2 comprises a deep liquid chromatography mass spectrometry
(LC/MS/MS)-based quantitative analysis of pooled samples (3 pools generated randomly from
each response cohort) from each of the identified response groups. Molecules that show
significant changes will be considered as candidate biomarkers of drug response. In Stage 3
immunoassays (ELISA) will be developed in order to test the validity of each novel biomarker
in its ability separately or in combination with existing biomarkers to predict drug
response.
Immune response: Whole blood samples will be incubated with Mu antigens overnight at 37
degrees celsius and the supernate will be stored for cytokine assays using ELISA as in our
previous studies (4).
Antigen stimulated T cell populations will also be studied by flow cytometry to assess the
proportion of Interferon(IFN) gamma, Tumor necrosis factor (TNF) and interleukin-2 (IL2)
secreting T cells in patients compared to controls.
Mycolactone detection: Lipids will be extracted from homogenised skin biopsies and
swabs//fine needle aspirates and the biological activity of mycolactone will be measured by
a cytotoxicity assay as described previously using human embryonic lung fibroblasts as
target cells. The presence of mycolactone in tissue samples will be confirmed and quantified
by mass spectrometry as previously described (5). Mycolactone production by the strain of Mu
isolated from oedematous lesions will be quantified in vitro and the tissue concentration of
mycolactone in oedematous lesions will be measured.
Sample size and justification There will be 450 participants in this study. Our previous
studies showed a mean difference in TNF alpha at baseline between fast and slow responders
to be 15pg/ml and a standard deviation of 33pg/ml. The standardized mean difference computed
as d=15/33 was 0.45. The sample size required to detect this difference with a two-sided
significance of 5% and with 80% power would be 100 participants in each group (that 200
patients). We expect an attrition rate of 20% bringing the sample size to 250 patients.
There will be control group of 200 healthy volunteers
Statistical analysis For the purpose of this study a "rapid responder or fast responder" is
defined as a patient with a time to healing of less than 12 weeks or rate of healing of
greater than 4mm/week and " slow responder or slow healer" is defined as a patient with time
to healing of 12 or more weeks or rate of healing of less than 3 mm/week
Data generated by multianalyte profiling will be analyzed by multivariate analysis,
including principal component analysis (PCA) and partial least-squares (PLS)-related
methods, using the SIMCA 13 software (Umetrics, Sweden) . Univariate analyses will be
performed to further characterize candidate biomarkers. Here, differences between controls
and disease patients will be analyzed using the non-parametric Wilcoxon rank sum
(Mann-Whitney) test. Paired samples for BUD patients at week 0 and week 12 will be compared
using the Wilcoxon signed rank test. Univariate analysis and multiple testing adjustments
will be performed using R software (version 2.13.2, R Development Core Team, R Foundation
for Statistical Computing, Vienna, Austria) and package QVALUE. GraphPad Prism software will
be used for graphical representation.
