Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT03439592 |
| Other study ID # |
142002018 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
January 1, 2016 |
| Est. completion date |
June 1, 2021 |
Study information
| Verified date |
October 2020 |
| Source |
University of Campania "Luigi Vanvitelli" |
| Contact |
Raffaele Marfella, MD, PhD |
| Phone |
+39 0815665110 |
| Email |
raffaele.marfella[@]unicampania.it |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Hyperglycemia is a common finding in patients diagnosed with acute coronary syndrome (ACS),
and an independent predictor of mortality in patients with and without diabetes. Though
percutaneous coronary intervention (PCI) is the cornerstone of ST-segment elevation
myocardial infarction (STEMI), the incidence of heart failure, re-infarction and death in
hyperglycemic patients remains significant, with a mortality of more than 40% one year after
the event. In these STEMI patients dual anti-aggregation therapy is currently the gold
standard after PCI, but bleeding phenomena, and therapeutic resistance may reduce their
therapeutic efficacy. Therefore, it is likely that the individual response to the dual
anti-aggregation therapy, and the hyperglycemic stress, may influence resistance mechanisms,
and/or lead to an increase in pharmacological functional deactivation by the microbiotic
flora. The term microbiota indicates the totality of the genomes of microorganisms that
reside in an ecological niche, and which constitute the "human microbiota". In this context,
the analysis of the faecal microbiota before PCI, at hospital discharge and at follow-up,
could be considered useful for identifying hyperglycaemic patients with alteration of
metabolic-oxidative processes, and pro-thrombotic correlates with worse post procedural
prognosis. Therefore, the analysis of faecal microbiota during the STEMI event could
theoretically identify hyperglycemic patients with excessive inflammatory and oxidative tone
caused by hyperglycemia, conditioning resistance to double anti-aggregation therapy and
coronary stenting, and conditioning pro-thrombotic phenomena after coronary reperfusion by
PCI. Therefore, authors will conduct a study to analyze the microbiota in patients with acute
hyperglycaemic and normoglycemic coronary syndrome. The primary objective of this study will
be to evaluate any changes in the microbiota and its activity on faecal material taken before
PCI, and after 6 and 12 months in patients with hyperglycemic STEMI, and also evaluate if the
changes in the microbiota can be related to the 12-month prognosis.
Description:
Hyperglycemia is a common finding in patients diagnosed with acute coronary syndrome (ACS)
and is an independent predictor of mortality in patients with and without diabetes (1-4).
Though percutaneous coronary intervention (PCI) is the cornerstone of ST-segment elevation
myocardial infarction (STEMI) (4-6), the incidence of heart failure, re-infarction and death
in hyperglycemic patients remains significant, with a mortality of more than 40% one year
after the event (4-5). In fact, restenosis and no-reflow phenomena are common events leading
to worse clinical outcomes, in hyperglycemic patients undergoing primary PCI, and resulting
in atherothrombotic embolization, increased oxidative stress and inflammation (3-6). In an
attempt to counter the increasing in mortality from hyperglycemia, the coronary units
implemented insulin protocols to normalize blood glucose during ACS, and the same insulin
treatment showed conflicting results (7). Data from small pilot studies, from moderate
clinical trials and a meta-analysis suggested benefits from insulin therapy (1-3.5). On the
other hand, subsequent randomized clinical trials have not confirmed an increase in survival
(7, 8) . Moreover, if the dual anti-aggregation therapy is currently the therapeutic basis of
patients with STEMI treated by PCI (9), increasingly frequent anti-aggregation therapy and
bleeding phenomena, may reduce their therapeutic efficacy (11 -13). Therefore, it is likely
that the individual response to the dual anti-aggregation therapy, and the hyperglycemic
stress, may influence resistance mechanisms, and/or lead to an increase in pharmacological
functional deactivation by the microbiotic flora. The term microbiota indicates the totality
of the genomes of microorganisms that reside in an ecological niche, and which constitute the
"human microbiota" (14). According to the analyzed anatomical district we speak of
intestinal, ocular, buccal, vaginal microbiota, etc. The DNA analysis of microorganisms
living in the human intestinal tract, realized with metagenomic methods by the MetaHIT
consortium, has identified over 3 million genes, 150 times those of the human species
(16-21). In addition, cardiovascular diseases such as hypertension and coronary artery
disease are strongly conditioned by the presence of Enterobacteriaceae, Firmicutes and
Lattobacilli in the intestine, which can determine their onset (22). In this context, the
analysis of the faecal microbiota before PCI, at hospital discharge and at follow-up, could
be considered useful for identifying hyperglycaemic patients with alteration of
metabolic-oxidative processes, and pro-thrombotic correlates with worse post procedural
prognosis ( 23). The ease of obtaining samples of faecal microbiota, and the relevant impact
on the scientific community, and on routine clinical practice, pushes us to insist on
clinical research, together with a genomic, metabolic and cellular analysis of the
microbiota, and to extract as much information as possible in hyperglycemic patients with
STEMI. This investigation has never been proposed in the scientific research and clinical
practice of hospitalized hyperglycaemic patients for STEMI. Indeed, a large amount of
evidence suggests that, hyperglycaemia causes an increasing production of oxidative stress
and inflammation, both in atherosclerotic plaque and in the thrombus, and therefore it plays
a central role in determining worse outcomes in STEMI. Therefore, the analysis of faecal
microbiota during the event could theoretically identify hyperglycemic patients with
excessive inflammatory and oxidative tone caused by hyperglycemia, conditioning resistance to
double anti-aggregation therapy and coronary stenting, and conditioning pro-thrombotic
phenomena after coronary reperfusion by PCI. Therefore, authors will conduct a study to
analyze the microbiota in patients with acute hyperglycaemic and normoglycemic coronary
syndrome.
OBJECTIVES The primary objective of this study will be to evaluate any changes in the
microbiota and its activity on faecal material taken before PCI, and after 6 and 12 months in
patients with hyperglycemic STEMI, and also evaluate if the changes in the microbiota can be
related to the 12-month prognosis.
MATERIALS AND METHODS Patients This is a cohort study to investigate the effects of possible
changes in the microbiota and its activity in faecal samples taken from patients with
hyperglycemic STEMI v/s normoglycemic. These patients are treated according to routine "real
life" therapeutic practices, adopted at the Cardiology Division of the "Luigi Vanvitelli"
University of Campania. After discharge from the hospital, all patients will be invited to
carry out the control visits, as indicated by the guidelines for the management of post-STEMI
patients (6), at the Cardiology Division of the University of Campania "Luigi Vanvitelli "and
at the VI Division of Internal Medicine of the" Luigi Vanvitelli "University of Campania. All
patients will be monitored for 12 months after the event, as a follow-up. According to the
recent definition of the American Heart Association, hyperglycemia will be defined by fasting
blood glycemic levels> 140 mg / dl (1, 9). All patients with onset of symptoms within 12
hours and 1 mm elevation of the ST segment in 2 or more contiguous peripheral leads or at
least 2 mm in 2 or more contiguous precordial leads or new onset left branch block will be
considered for PCI. Inclusion criteria will include: age over 18 and admission for STEMI
first episode. Patients with left ventricular ejection fraction <25%, with previous IMA or
previous PCI and / or by-pass will be excluded. Each will be included in the study after
signing an informed consent. Routine haematological analysis will be performed at the time of
acceptance, before practicing medical therapy and possible coronary artery examination and
after 6 and 12 months. On an aliquot of the blood sample taken at these times, we will
evaluate trimethylamine-N-oxide (TMAO), a product of bacterial metabolism, whose elevated
serum levels are associated with adverse cardiovascular events (24). In addition, before the
PCI, and 6 and 12 months later, a stool sample will be taken on which the microbiota will be
subsequently analyzed. The research will be conducted in accordance with the principles of
the Helsinki declaration.
Procedure In this study authors will evaluate a cohort of patients with STEMI admitted to the
Cardiology Division of the "Luigi Vanvitelli" University of Campania, and treated by dual
anti-aggregating therapy. All patients will be treated according to the suggested protocols
of the latest Guidelines on STEMI (6). Patients will be classified into two groups:
hyperglycaemic patients v/s normoglycemic, in both groups of patients will be carried out
analysis of the fecal microbiota, pre PCI and pre dual antiplatelet therapy. The microbiota
will be extracted from faecal material, and then prepared and subsequently analyzed. It is
not possible in clinical practice to culture in the laboratory the vast majority of
intestinal bacteria, as compared to the enormous amount of microorganisms present. Thanks to
the most up-to-date techniques of sequencing the bacterial DNA (Next Generation Sequencing)
extracted from the faecal material, it is now possible a complete and reliable identification
of the intestinal microbiota. In addition to identify the bacterial species present, authors
are also able to express an overall health degree of the microbiota (or dysbiosis), its
efficiency in the production of some useful or harmful substances, the adequacy of the
microbiota with respect to some fundamental functions to which is a deputy and the propensity
of the microbiota itself against inflammatory bowel diseases, metabolic diseases and aging.
The result is an impressive amount of data, a true identity card of the microbiota, named the
Microbiota passport.
Sample preparation and analysis. Samples for sequencing the microbiota will be collected from
faecal material and stored at ultra-low temperature (-80°C) to avoid damage to the original
sample. The sequencing of the microbiota will be performed by the laboratory of Microbiology
of the University of Campania "Luigi Vanvitelli". For the sequencing of the microbiota we
will use the "NextGeneration Sequencing" (NGS) method, which allows the sequencing in
parallel of millions of DNA fragments (25) through the use of Illumina machinery (San Diego,
CA). The DNA will be extracted from these samples using a high reproducibility commercial kit
procedure (Qiagen, Invitrogen). The final yield and quality of the extracted DNA will be
determined using the NanoDrop ND-1000 spectrophotometer (ThermoScientific, Waltham, MA) and
QubitFluorometer 1.0 (Invitrogen Co., Carlsbad, CA). The extracted DNA will be amplified by
PCR with the primers: forward: 5'-CCTACGGGNGGCWGCAG-3 'and reverse:
5'-GACTACHVGGGTATCTAATCC-3' (Klindworth et al.2013), which target the hypervariable region V3
and V4 of the 16S rRNA gene . Each PCR amplification will be performed by protocol for the
preparation of 16S libraries for metagenomic sequencing (Illumina, San Diego, CA). The
amplification products will be quantified by the Qubit fluoridator (Invitrogen Co., Carlsbad,
CA) and grouped at an equimolar amount of each sample with a final concentration of 2nM. A
Phix control library (Illumina) will be added to the samples. The grouped samples will then
be sequenced on the MiSeq Platform (Illumina, San Diego, CA). The data thus obtained will be
subjected to quality controls by bioinformatics analysis with FastQC. Finally, the data will
be analyzed according to the statistical tests suggested by the guidelines for metagenomic
sequencing. Patient data will be compared with each other and over time with reference
databases. The evaluation of the epigenetic activity of the microbiota and the biochemical
dosages of cytokines and free radicals will be performed at the laboratory of Pharmacology of
the University of Campania "Luigi Vanvitelli. In particular, for the evaluation of the
epigenetic activity of the microbiota of each individual hyperglycemic v/s normoglycemic
patient with STEMI, we will proceed according to the protocol QIAGEN (Italy) with the
extraction from faecal material of the total RNA, including the low-weight one molecular
(microRNA). The final yield and quality of the extracted RNA will be determined using the
NanoDrop ND-1000 spectrophotometer (ThermoScientific, Waltham, MA). Through qRT-PCR (QIAGEN,
Italy) the expression levels of the following fecal microRNAs will be detected: miR-10b,
miR-204, miR-29, miR-496, miR-1224-5p, miR-470 and miR-663 involved in the regulation of the
intestinal microbiota; miR-10a and miR-18b involved in the regulation of intestinal
microbiota and metabolic inflammation; miR-106a, miR-17, miR-19a, miR-20a, miR-206, miR-222
and miR-223 involved in metabolic inflammation and signaling insulin/IGF-1 (26). The
metabolic activity of the microbiota in each individual hyperglycaemic v/s normoglycemic
patient with STEMI will be evaluated by determination of short chain fatty acids (SCFA) from
faecal samples: the levels of fecal SCFA, a product of bacterial fermentation, seem to
correlate with an improvement in type 2 diabetes (27). The inflammatory activity of the
microbiota will be evaluated by determining the fecal levels of LPS (28-29), IL-1β and IL-6
(30-32), while the oxidative activity through the analysis of fecal levels of ROS, SOD and
GSH (33-34).
Follow-up Authors' study is designed to manage hyperglycaemic v/s normoglycemic patients with
STEMI in the "real life", without interference from the rigid rules that limit the
generalisability of RCTs. Therefore, after discharge from the hospital, all patients will be
invited to carry out the control visits, as indicated by the guidelines for the management of
post-STEMI patients (6), at the Cardiology Division of the University of Campania "Luigi
Vanvitelli", and at the VI Division of Internal Medicine of the "Luigi Vanvitelli" University
of Campania. All patients will be monitored for 12 months after the event, as follow-up, to
practice clinical evaluation (ECG, exercise test, echocardiogram, glucose levels), as
indicated by the guidelines for the management of STEMI patients (6). At these time points,
stool samples will also be taken for the analysis of the microbiota. Moreover, in the
management and assessment of the glycemic compensation in the 12 months of follow-up, the
physician will also be involved in order to maintain HbA1c levels <7%, glycemia between 90
and 140 mg / dl and postprandial glycemia <180 mg / dl.
Cardiovascular endpoints The cardiovascular end points in both cohorts will include
myocardial re-infarction, re-hospitalization for coronary artery disease (re-stenosis, stent
implantation or aorto-coronary bypass), heart failure, stroke, cardiac mortality, and all
causes of mortality. The primary composite end point will consist of cardiovascular events,
such as myocardial infarction, hospitalization for heart failure, stroke or cardiac
mortality. The secondary end point will consist in the evaluation of cardiac function:
reduction of ejection fraction, increase in volume of the left ventricle, reduction of
coronary perfusion. All deaths will be reviewed and classified as cardiac (death caused by
IMA, ventricular arrhythmias, refractory heart failure) or non-cardiac. All
revascularizations will be classified as early (elective revascularization within 60 days
after coronary angiography) or late. Only late revascularizations will be classified as
cardiac events, so patients with early elective revascularization will be excluded from the
analysis.
Statistical analysis The two groups will be compared using the Pearson χ2 test for
categorical variables and the Kruskal-Wallis test for continuous variables. The covariate
candidates for entry into the multivariable model will be identified by concentrating on the
factors those that will significantly differ (P value <0.05) in the univariate analysis
between hyperglycemic v/s normoglycemic patients. Cox regression will be used to build the
mortality model. The Hazard Ratio for mortality will be adjusted for age, BMI, cholesterol,
LDL, triglycerides and aspirin therapy, ticlopidine, combination of anti-aggregants,
β-blockers, ACE inhibitors or sartans, antidiabetics, statins, during hospitalization for
STEMI. Survival analysis through the first year from STEMI will be performed using the
Kaplan-Meier curve and Cox regression method. The mortality curves will be obtained
separately for hyperglycaemic patients v/s normoglycemia, to be compared using the log-rank
test. All tests will be considered significant if p value <0.05. All analyzes will be carried
out in the two study populations. A "propensity score" analysis will be performed using a
"non parsimonious" logistic regression model that will compare the outcomes of the
hyperglycemic v / s normoglycemic patients. More variables will be included in the model
including age, sex, diabetes, hypertension, hypercholesterolemia, multivessel disease,
chronic renal failure, TIMI pre-procedure flow, ejection fraction and procedural success. The
C-score will indicate good discrimination. Based on the "matched" samples, the Cox
proportional risk model will be used to determine the impact of hyperglycemia, and of
microbiotic flora activity on mortality during follow-up. SPSS (version 21, IBM SPSS) will be
used for all analyzes.
Sample Size The Sample Size will be calculated with IBM PC computer by GPOWER software. The
size effect will be calculated according to the epidemiological and interventional studies
performed on hyperglycaemic patients. The sample size estimated on a global effect of 25%
with type I error of 0.05 and a power of 95% will be 200 participants, 100 for the group
composed of normoglycemic patients and 100 for the group of hyperglycaemic patients.
