Evolution of myocardial oedema and fibrosis in HIV infected persons after the initiation of antiretroviral therapy: a prospective cardiovascular magnetic resonance study

Detailed methodology regarding the recruitment process of the project have been published elsewhere [21] and this cohort has subsequently been expanded in this study. In short: Newly diagnosed, HIV infected individuals were recruited into a prospective cohort study in the Western Cape, South Africa. Participants were recruited as out-patients as they tested positive for HIV, along with a healthy, HIV uninfected control group from the same areas, frequency matched for age and sex. The HIV status of all persons were confirmed with a serum HIV enzyme-linked immunosorbent assay and eligible participants were enrolled before the initiation on first line ART (tenofovir/lamivudine/dolutegravir). The ART naïve status of participants was cross checked on a centralised pharmacy system. All participants recruited after March 2020 underwent severe acute respiratory syndrome coronavirus-2 polyumerace chain reaction (PCR) testing before inclusion. All patients aged 18 to 55 years, without known current or prior cardiac disease and without ART use prior to enrolment, who were not pregnant or acutely unwell (including current coronavirus disease-19), were deemed eligible for inclusion. Potential participants with a contraindication to CMR or gadolinium contrast, were excluded. Participants diagnosed with tuberculosis (TB) coinfection, were not excluded. HIV/TB coinfected participants were enrolled at least 2 weeks after initiation on TB treatment while still ART naïve. All treatment and workup provided to HIV infected participants were in line with South African treatment guidelines [22].

Data were collected at three time points for HIV infected participants: A baseline visit prior to ART, an interim visit at 4 months, and a final follow-up after at least 9 months on ART. The HIV uninfected healthy subjects had a once-off research visit. Full evaluation including anthropometric, biochemical, immunological, virological, electrocardiogram (ECG), and CMR investigations were performed at baseline and final follow up. Additional CD4 count and HIV viral load measurements were made at the interim visit. Participants were seen in the morning following a fast of at least 10 h. The World Health Organisation (WHO) HIV clinical stage was documented [23]. Blood pressure was measured in the seated position after a 10-min rest on the same wall-mounted sphygmomanometer unit (Welch Allyn, New York, USA). Mean arterial pressure was calculated as [(1/3 × pulse pressure) + diastolic blood pressure] [24]. Height and weight were measured, and body mass index (BMI) was calculated. Waist circumference was measured standing at the level of the umbilicus. A 12-lead ECG was performed. A 6 min walk test (6MWT) was recorded using standardised methodology [25]. Fasting blood samples were analysed by the on-site National Health Laboratory Service for measurement of urea and electrolytes, glucose, blood lipids, full blood count, differential cell count, HIV enzyme-linked immunosorbent assay, high sensitivity C-reactive protein (hsCRP), flow cytometry based CD4- and CD8 count, and HIV-1 viral load. The linear range for the measurement of HIV viral load by our core laboratory is 20 to 10,000,000 copies/ml or 1.3 to 7 log (Abbot Alinity M HIV-1 assay, Abbott Park, Illinois, USA). We defined persistent viraemia as a viral load of > 200 copies/ml in persons on ART at interim follow-up or at 9 months in accordance with contemporary research [26]. Gender-specific estimated glomerular filtration rate (eGFR) was calculated using the Cockcroft–Gault formula [22]. At the final visit, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G antibodies were tested in the HIV group.

Sample size calculation was performed for CMR-measured LV volumes, mass, and ejection fraction (LVEF) using paired samples t-test for the prospective evaluation of the HIV infected group. Statistical analyses were performed using SPSS (version 27, Statistical Package for the Social Sciences, International Business Machines, Inc., Armonk, New York, USA) and STATA (version 17.0, Stata Corporation, College Station, Texas, USA). Continuous data are presented as mean ± standard deviation. For non-normally distributed data, the median and interquartile ranges are stated. Categorical data is presented as frequencies with percentages. The normality of data was assessed using the Shapiro–Wilk test. For cross sectional analyses between the HIV infected and control group, the Chi-square test, independent samples t-test, and Mann–Whitney U tests were used as appropriate. For prospective analyses of the HIV infected group, the paired samples t-test, Wilcoxon signed-rank test, or McNemar test was employed as appropriate. Inter-reader reliability on quantitative data was assessed using interclass correlation coefficient (ICC). A two-way random, average measurements with absolute agreement model was used. Cohen’s Kappa was used to evaluate the agreement of categorical variables between readers. Bivariate correlations were calculated using Spearman’s Rho. Multivariate analysis with native T1 as outcome variable was performed with quantile regression models. Statistical significance was 2-tailed and defined as a p-value ≤ 0.05.

All participants that underwent a 9-months follow up CMR were included in the study (Fig. 1). The study group’s demographics and clinical data are shown in Table 1. The HIV infected group and healthy controls were well matched in terms of age and sex. Both groups were relatively young with a mean age of 33 and 32 years, respectively. The income of the HIV infected group was considerably lower compared with controls, and reflect the lower socio-economic status of this population. Although the HIV infected group had more smokers (49% vs 27% in the control group), this did not reach statistical significance. Ethanol use showed a wide spread in the data, but proved statistically similar in the naïve group and controls. 97% of the cohort a dolutegravir-based regimen. After initiation on ART, participants had the tendency to consume considerably less alcohol. Chronic disease and medication use were rare, and comparable between the controls and HIV infected group. Two HIV infected participants had a known diagnosis of hypertension. During follow up, one HIV infected participant developed hypertension and received treatment. Metabolic syndrome was rare with only one control and two participants fulfilling the National Cholesterol Education Program Adult Treatment Panel III [30] criteria at enrolment.

The median time from enrolment to repeat CMR evaluation was 9 months (Interquartile range: 9 to 13 months).

Sixty six percent of the cohort had early (WHO I or stage II) disease. A third of the participants visited their health centre due to significant, unexplained weight loss and were otherwise well. The remainder of the HIV group presented due to contact tracing or with HIV related complications. These included dermatological manifestations of HIV, lymphadenopathy, respiratory-, gastrointestinal-, and miscellaneous complaints. HIV/TB coinfection was found to be prevalent among newly diagnosed people living with HIV. Ten naïve participants (14% of the HIV group) had TB disease diagnosed around the time of enrolment. All but one of the TB cases were mild and managed on an out-patient basis. Extrapulmonary TB was diagnosed incidentally in 2 cases at baseline (abdominal and pleural TB respectively). The 6MWT demonstrated a comparable functional status between the naïve group and controls (618 vs 637 m, p = 0.4). No HIV infected participant reported any significant loss of functional capacity or dyspnoea. Furthermore, ART did not cause a significant improvement in the 6MWT.

Biochemical and immunological data are shown in Table 2. At 9 months on ART, participants had a median viral load of 20 copies/ml (Interquartile range: 20 to 36). Median hsCRP measured 50% higher in the naïve group than controls (3.6 vs 2.4 mg/l). This did however not reach statistical significance (p = 0.5). A marked decrease in the median hsCRP was observed when comparing the HIV infected participants at enrolment and 9-month follow-up (3.4 to 1.9 mg/l, p = 0.01).

CMR data are shown in Table 3. The presence of small (< 10 mm), haemodynamically insignificant pericardial effusions were highly prevalent in the naïve group compared with controls (67% vs 18%, p < 0.001). No study participant had pericardial LGE. The pericardial effusions were observed in the absence of pericardial thickening and were not associated with the presence of TB disease.

Although a trend of abnormality was observed, global T2 SIR did not show a statistical difference between the naïve group and controls (p = 0.5). When evaluating the HIV group over time, a trend of increasing T2 SIR, most notable at the basal segments, was observed. This however did not reach our study’s requirements for significance (p = 0.08). EGE SIR did not demonstrate any difference amongst the study groups.

The segmental native T1 times of the study groups are shown in Fig. 2. Both the global native T1 and T2 mapping values were higher in the naïve group compared with controls. These differences were most notable at the heart base with mean differences of 23 ms (ms) and 2 ms for global native T1 and T2 respectively. The mean global ECV of the control group measured 2% lower compared with the naïve group. Only a small number of controls received contrast, and this did not reach statistical significance when compared to the ART naïve group (p = 0.1). However, global native T1 (obtained in all controls), demonstrated a high linear correlation with ECV across all study groups (r_s = 0.8, p < 0.001).

After ART initiation, significant changes in the HIV group’s mapping were observed. The global native T1 and ECV showed significant change towards normalisation and were statistically similar at 9 months when compared to the control group (Table 3 and Fig. 3). No significant change was observed in the global T2 after ART initiation.

LGE was 5 times more prevalent in the HIV infected group compared with the control group (49% vs 10%, p = 0.02). Although unexpected, one control had LGE in keeping with non-ischaemic replacement fibrosis. In the HIV group, more than two thirds of visualised LGE was present in the basal segments and most frequently involved the inferoposterior walls. The pattern of LGE was predominantly subepicardial, with a smaller number of participants demonstrating midmyocardial enhancement, or a combination of the two. In all cases where LGE was visualised at baseline, LGE was seen at 9 months as well. Two cases were thought to have de-novo LGE at follow up. Three cases had LGE at follow up that did not meet study criteria of LGE at the initial evaluation.

Fifteen HIV infected persons demonstrated persistent viraemia and this group’s study parameters are shown in Table 4. All these cases had a history of poor compliance on ART preceding follow up. Although not proven to be significant, a marginal increase in pericardial effusion prevalence was observed over time (67% to 73%, p = 0.7).

The persistent viraemia group’s global native T1 did not decrease to the same degree as the greater HIV cohort and differences are evident when compared with the virally supressed participants (n = 58) that represent 79% of the HIV infected cohort (see Table 5). No significant difference in global native T1 was evident after 9 months on ART in the virally unsuppressed group (p = 0.3). The virally supressed subgroup demonstrated a threefold decrease in the global native T1 time compared with the persistent viraemia subgroup (the mean decrease in T1 of these two subgroups at recruitment and 9 months were 21 ms and 7 ms respectively). Furthermore, no significant difference in the global ECV of the persistent viraemia group was seen over time as was seen with virally supressed individuals. The global T2 in the virally unsuppressed subgroup increased by one ms, although this did not prove to be statistically significant.

The quantile regression model for global native T1 is shown in Fig. 4. The HIV viral load (log) was significantly associated with the global native T1 at the time of HIV diagnosis (p = 0.02) when adjusted for age, sex, tobacco, BMI, and CD4 count.

The re-read of multiparametric tissue characteristics demonstrated good to excellent ICC between readers. The quantitative parameters were analysed, and their correlations included: Global native T1 (ICC = 0.85), Global T2 (ICC = 0.95), global T2 SIR (ICC = 0.86), EGE SIR (ICC = 0.82), and ECV (ICC = 0.98). Probability values were all calculated to be highly significant (p < 0.001). Excellent inter-rater reliability was shown for the identification of pericardial effusion (Kappa = 1.0, p < 0.001) and qualitative assessment of LGE (Kappa = 0.86, p < 0.001).

Myocardial fibrosis and/or oedema may explain the raised native T1 observed in our HIV infected group [10, 32]. Native T1 measurement allows for the uncontrasted assessment of a variety of pathological processes (ranging from subclinical to end-stage disease) in both the myocyte (intracellular compartment) and the interstitium (extracellular compartment) [33]. Although changes are not specific to a particular disease process, native T1 mapping is a sensitive tool to assess pathology related to oedema, necrosis, fibrosis, protein-, lipid-, and iron deposition [33] and is superior to LGE and T2-weighted imaging to detect myocardial inflammation [34, 35]. In addition to focal pathology, T1 mapping is able to provide information on diffuse myocardial disease and has distinct technical advantages over LGE imaging [36].

Notably, our mapping methodology included an endocardial and epicardial offset to increase both accuracy and reproducibility of measurements of the midmyocardium. Consequently, the subepicardial abnormalities visualised on LGE-imaging are not fully reflected in the native T1 measurements and these two parameters should be considered independently. In accordance with our study hypothesis, a decrease in the native T1 time was identified in the HIV infected group after initiation of ART. The notion that the native T1 decrease was due to the reversal of diffuse myocardial fibrosis is controversial and less likely here [37]. Our findings are better explained by the improvement of subclinical myocardial oedema secondary to ART initiation as the decrease in native T1 was associated with a decrease in HIV viral load, hsCRP, and an increase in CD4 count. Furthermore, the ventricles of our HIV infected cohort were heavier compared with controls, in keeping with observations from prior research [10, 21, 38]. Although not conclusive, we have demonstrated signal that the native T1 decrease in persons with suboptimal compliance and persistent viraemia do not show the same degree of native T1 decrease. These findings all support oedema as the cause for the native T1 findings in the study.

Increased signal in T2-weighted imaging is specific, although less sensitive, for detecting increased absolute, as well as free intracellular water in the myocardium [39]. The updated Lake Louise Criteria have incorporated T1- and T2 mapping abnormalities as main criteria to diagnose myocardial inflammation [35]. The presence of raised T2 mapping values as seen in our HIV infected cohort, coupled with high native T1 times and a high prevalence of pericardial effusion, provides further support for the presence of myocardial inflammation. The observation that the T2 mapping values did not decrease concurrently with T1 mapping, may be explained by the superior sensitivity of T1 to detect myocardial tissue abnormalities. As T2 mapping has excellent specificity to detect myocardial oedema, this provides evidence for the incomplete resolution of chronic myocardial oedema in the HIV group. This finding is in keeping with CMR research from a cohort of ART experienced people living with HIV, that demonstrated elevated T1 and T2 despite viral suppression [26].

Our ECV data is also in keeping with interstitial expansion secondary to extracellular oedema at the time of HIV diagnosis, with subsequent improvement on ART at 9 months. ECV has the ability to detect milder interstitial expansion not readily detectable by LGE and predicts clinical outcomes that has been shown to be as important as LVEF when estimating prognosis [40, 41]. The decrease in the ECV seen at 9 months in our data is likely indicative of improved cardiovascular risk for PLWH, but will require formal evaluation in outcome studies.

The focal LGE in our cohort is in keeping with non-ischaemic, replacement type fibrosis (Fig. 5). In accordance with the largest LGE study in people living with HIV to date, the prevalence of fibrosis in our cohort was significantly higher in the HIV infected group compared with controls [42]. LGE did not change significantly over time, further supporting the presence of myocardial fibrosis. Comparing our LGE findings with published data in ART naïve persons from a low- to middle income country, the prevalence and pattern of LGE do not differ significantly (49% vs 39% between studies with predominantly subepicardial and midmyocardial LGE reported) [12]. A study from a high income country has reported LGE in as many as 85% of ART naïve individuals, although this study only evaluated 13 ART naïve persons [10].

Our findings support the growing body of evidence that, in addition to myocardial oedema, a component of replacement-type, myocardial fibrosis may be present in people living with HIV [10, 12]. The long- term significance of this finding, already demonstrable at the time of HIV diagnosis, is unknown. Although diffuse myocardial fibrosis have been linked to poor outcomes in persons with heart failure [43], the long term implications of fibrosis seen in people living with HIV require further study. An autopsy study that evaluated HIV infected persons with sudden cardiac death found that total fibrosis (interstitial-, perivascular-, and replacement fibrosis) was on average 72% higher in people living with HIV compared to HIV uninfected persons [32]. Although a higher rate of sudden cardiac death attributable to arrythmia was not conclusively shown, the reported incidence rate ratio of 1.87 suggests this is possible. Longitudinal studies evaluating the influence of different types of myocardial fibrosis on prognosis are lacking.

ART itself may have deleterious effects on cardiovascular health and is another mechanism of CVD in people living with HIV. Our cohort was on a dolutegravir-based ART regimen and unfortunately little is known on the long term cardiovascular effects of integrase inhibitors. Integrase inhibitors are thought to be have a more favourable cardiovascular side effect profile compared with protease inhibitors that have well-described pro-atherogenic effects [44]. However, a recent publication has shown a possible link with myocardial infarction, stroke, and cardiovascular intervention [45]. ART side effects with a co-existing subclinical cardiomyopathy remains unstudied. Our study explored inadequate viral suppression as a mechanism for CVD and not specifically the type of ART, as most of our participants received the same regimen. Therefore our results cannot necessarily be extrapolated to all ART regimens.