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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 29, No 3, May/June 2018
AFRICA
151
A final number of 91 patients with presumed CRMR
underwent clinical evaluation, resting electrocardiogram
and detailed echocardiographic assessments according to a
pre-determined protocol. Of these 91 patients with CRMR,
69 were excluded due to the following: co-morbidities (human
immunodeficiency virus:
n
=
22; hypertension:
n
=
44; diabetes
mellitus:
n
=
3; atrial fibrillation:
n
=
4; anaemia:
n
=
3; renal
dysfunction:
n
=
3; and inadequate image quality:
n
=
5).
The final sample comprised 22 patients. Fourteen age- and
gender-matched controls were also enrolled for the biomarker
arm of the study. A tolerance of five years was allowed for age
matching.The baseline clinical characteristics of these individuals
were recorded and they subsequently underwent comprehensive
echocardiography and CMR imaging.
The studywas approvedby theUniversityof theWitwatersrand
ethics committee. It was conducted in accordance with the
principles outlined in the Declaration of Helsinki.
Transthoracic echocardiography was performed on all patients
in the left lateral position by experienced sonographers using a
S5-1 transducer on a Philips iE33 system (Amsterdam, the
Netherlands). Images were obtained according to a standardised
protocol. The data were transferred and analysed off-line using
the Xcelera workstation.
All linear chamber measurements were performed according
to the American Society of Echocardiography (ASE) chamber
guidelines.
17
Measurements relating to LV diastolic function were
performed in accordance with the ASE guidelines on diastolic
function, and included pulse-wave Doppler at the mitral tips and
tissue Doppler of both medial and lateral mitral annuli.
18
MR was considered rheumatic in aetiology when the
morphology of the valve satisfied the World Heart Federation
(WHF) criteria for the diagnosis of chronic rheumatic heart
disease (RHD).
19
MR severity was assessed using qualitative,
semi-quantitative and quantitative methods as per the ASE
and European Society of Cardiology.
20,21
In equivocal cases,
the echocardiographic data were integrated with the clinical
evaluation by an experienced cardiologist to distinguish moderate
from severe MR.
For biomarker analysis, peripheral venous blood samples were
drawn from 14 controls and 22 chronic rheumatic heart disease
subjects at the time of echocardiographic examination. Samples
were collected in a serum separator tube and allowed a clotting
time of 30 min before centrifuging for 15 min at 1 000
g
and the
serum was stored at –80°C before analysis in a single batch.
Enzyme-linked immunosorbent assays (ELISA) were used
to determine the serum concentration of PIIINP (USCN
Life Science Inc/Cloud-Clone Corp, Wuhan, China), and PIP
(Clontech,Takara Bio Inc, Japan) was determined by ELISA
using the USCN kit according to the manufacturer’s instructions.
The minimum detectable dose of PIIINP is typically less than
25.9 pg/ml and the minimum detectable dose of PIP is typically
less than 10 ng/ml. Optical density of the analytes was measured
at 450 nm on a microplate reader (Elx800, Biotek, USA) and
concentrations were determined using a 5-PL algorithm.
Multi-analyte analysis of the matrix metalloproteinase 1
(MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-
1) was performed using the magnetic luminex screening assay
(RnD systems, Minneapolis, USA) on a Bio-Plex 200 (Bio-Rad,
CA, USA) according to the manufacturer’s instructions. The
minimum detectable dose of MMP-1 is typically less than 2.7
pg/ml, and for TIMP-1, less than 3.42 pg/ml. Since the actual
activity of MMP-1 depends on the balance between active
enzyme and inhibitor (i.e. TIMP-1), the serum MMP-1/TIMP-1
ratio was considered an index of MMP-1 activity.
CMR studies were performed on a 1.5-Tesla scanner (Siemens
Healthcare, Erlagen, Germany) using a six-channel phased-
array body coil. The images were obtained during patient
expiratory breath-hold for approximately eight seconds and were
prospectively ECG gated.
22
LV volumes and mass were acquired
in line with standard cardiovascular MRI protocols (1.5-T
magnetom Avanto; Siemens Healthcare, Erlangen, Germany).
Steady-state free-precession imaging (echo times 1.5/3.0 ms, flip
angle 60°, temporal resolution 45 ms, slice thickness 7 mm, 3-mm
gap, matrix size 256
×
256 mm, field of view 380
×
309 mm) were
performed to obtain long-axis cinés and a contiguous stack of
short-axis cinés for assessment of LV volumes, mass and ejection
fraction, as previously described.
23
Ten minutes after the injection of 0.2 mmol/kg gadolinium-
based contrast agent (Magnevist, Schering, Berlin, Germany),
LGE-CMR images were acquired in the same long- and short-
axis position, and used in the ciné imaging.
5
Inversion recovery
times varying from 200–350 ms were used to null the signal from
the intact myocardium.
Images were analysed by an independent experienced reader
(RN), blinded to the echocardiographic results, with Argus
software (version 2002B, Siemens Medical Solutions, Erlangen),
as previously described.
24
The assessment of cardiac function
and chamber sizes were performed in standard views in the long-
axis (horizontal and vertical) and short-axis planes. Ejection
fraction for the LV was assessed with the following formula:
Ejection fraction
=
end-diastolic volume – end-systolic volume
_________________________________
end-diastolic volume
LV volumes and EF were obtained by semi-automatic tracing
of contours on the short-axis images in end-diastole and
end-systole, withmanual corrections when required.
25
Myocardial
fibrosis was defined as a region of LGE with signal enhancement
greater than the signal intensity of non-enhanced myocardium.
1
Statistical analysis
Statistical analysis was performed with Statistica version 12.5,
series 0414 for Windows. Continuous variables are expressed as
means
±
SD or medians (IQRs). Categorical data are expressed
as a percentage. The differences for continuous variables were
calculated using the Student’s
t
-test or Mann–Whitney
U
-test
when the distribution was non-normal. Chi-squared and Fisher’s
exact tests were used to calculate the difference for categorical data
for independent samples. Pearson’s and Spearman’s correlation
coefficient were used to calculate correlations depending on
whether data were normally or non-normally distributed.
Biomarker levels (TIMP-1, MMP-1 and MMP-1/TIMP-1 ratio)
were log transformed before analysis when distribution was not
normal. A
p
-value
<
0.05 was considered statistically significant.
Results
The mean age of patients was 36.3
±
13.9 years, with 81%
female (Table 1). All the patients had isolated moderate or severe
CRMR and no co-morbidities. Ten patients were in New York
heart association (NYHA) functional class I, the remainder