The association of atherosclerotic cardiovascular disease and statin use with inflammation and treatment outcomes in tuberculosis

Tuberculosis (TB) and atherosclerotic cardiovascular disease (ASCVD) have a close epidemiological and pathogenetic overlap. Thus, it becomes essential to understand the relationship between ASCVD and TB outcomes. From our retrospective cohort on drug-susceptible TB patients at the National Taiwan University Hospital, we assessed the association of pre-existing ASCVD (coronary artery disease (CAD) and atherothrombotic stroke (ATS)) with 9-month all-cause and infection-related mortality and the extent of mediation by systemic inflammatory markers. We determined the effect of pre-existing ASCVD on 2-month sputum microbiological status. Among ASCVD patients, we assessed the association of statin use on mortality. Nine-month all-cause mortality was higher in CAD patients with prior acute myocardial infarction (CAD+AMI+) (adjusted HR 2.01, 95%CI 1.38–3.00) and ATS patients (aHR 2.79, 95%CI 1.92–4.07) and similarly, for infection-related mortality was higher in CAD+AMI+ (aHR 1.95, 95%CI 1.17–3.24) and ATS (aHR 2.04, 95%CI 1.19–3.46) after adjusting for confounding factors. Pre-existing CAD (AMI- or AMI+) or ATS did not change sputum culture conversion or sputum smear AFB positivity at 2 months. The CAD+AMI+ group had significantly higher levels of CRP at TB diagnosis in the multivariable linear regression analysis (Adjusted B(SE) 1.24(0.62)). CRP mediated 66% (P = 0.048) and 25% (P = 0.033) of the association all-cause mortality with CAD+AMI− and CAD+AMI+, respectively. In summary, patients with ASCVD have higher hazards of 9-month all-cause and infection-related mortality, with elevated serum inflammation mediating one to three-quarters of this association when adjusted for confounders. Statin use was associated with lower all-cause mortality among patients with ASCVD.


Scientific Reports
| (2021) 11:15283 | https://doi.org/10.1038/s41598-021-94590-x www.nature.com/scientificreports/ Statins, apart from the role in primary and secondary CAD prevention, are known to reduce systemic inflammation 23 . Preclinical models have shown that statins enhance autophagy and phagosome maturation in Mycobacterium tuberculosis-infected macrophages and reduce the bacillary burden in human macrophages. As adjunctive therapy, statins improve sterilizing activities of the first-line anti-tubercular regimen in murine models [24][25][26][27] . Though systematic reviews have shown that statin use reduces the incidence of active TB 28,29 , there is no clinical evidence on whether statins have salutary effects in TB patients after adjusting for confounding factors 30 .
In our study, we assessed the association of pre-existing ASCVD with all-cause and infection-related mortality during the first nine months of TB treatment and evaluated to what extent the levels of systemic inflammatory markers mediate this association. We also determined the effect of pre-existing ASCVD on 2-month sputum culture and acid-fast bacilli (AFB) smear conversion. Among patients with pre-existing ASCVD, we assessed the association of statin use with 9-month mortality.

Methods
Study design and population. Our retrospective cohort included all consecutive adults (aged > 18 years) with culture-confirmed drug-susceptible TB, treated according to American Thoracic Society guidelines 31 at the National Taiwan University Hospital (NTUH), a referral center in Taipei city from 2000 to 2016, with no specific exclusion criteria 32 . All data were obtained from the NTUH database. The study was approved by the institutional review boards (IRB) at Johns Hopkins University and NTUH and the study methods were carried out in accordance with the IRB guidelines and regulations. As this was a retrospective chart review, informed consent from the study subjects was waived off by the IRBs at NTUH and Johns Hopkins School of Medicine.
Exposures. The three exposures assessed in our study are as follows: (1) CAD: the diagnosis of pre-existing CAD was based on either angiographic evidence of CAD, admission ICD codes, or review of outpatient medical records. For this exposure, the patients were stratified into three groups: No evidence of CAD (CAD-), CAD without history of AMI (CAD + AMI − ) and CAD with history of AMI (CAD + AMI + ); (2) Atherothrombotic stroke (ATS): The ATS subtype of stroke, based on admission ICD codes or outpatient medical records, was our exposure of interest; (3) Statin use among patients with ASCVD (CAD or ATS): For this study, we defined preexisting ASCVD as the presence of pre-existing CAD or ATS. Exposure for the "intention-to-treat" analysis for statin use was defined as the use of any of the following equivalent doses of moderate-intensity statin therapy 33 : atorvastatin 10 mg, simvastatin 20 mg, pravastatin 40 mg, rosuvastatin 10 mg, lovastatin 40 mg, fluvastatin 80 mg or pitavastatin 2 mg 33 for a minimum of 2 weeks (14 doses) in the first month of TB treatment. Exposure for the "per-protocol" analysis was defined as the use of any of the above doses of statins for > 80% (7.2 months) of the 9 months following the initiation of TB treatment, or > 80% of the duration of follow-up if lost to follow-up or death occurred before nine months.
Systemic inflammatory markers. C-reactive protein (CRP)(mg/dL), total leukocyte count (WBC) (× 10 3 /µL) and neutrophil-lymphocyte ratio (NL ratio) available at baseline were documented. Any test result for the inflammatory markers within 30 days of TB diagnosis was considered as baseline.
Outcomes. Primary outcomes were all-cause and infection-related mortality during the first 9 months of TB treatment. Infection-related mortality was a composite outcome of death due to pneumonia, sepsis, or TB. Secondary outcomes included sputum AFB smear positivity and culture positivity at 2 months after treatment initiation.
Statistical analysis. Patient characteristics, stratified into three groups of CAD status (non-CAD, CAD + AMI − , CAD + AMI + ), were compared using ANOVA for normally distributed data, Kruskal-Wallis test for non-normally distributed data, and chi-square (χ 2 ) test for categorical variables. Patient characteristics stratified by the presence of ATS, the second exposure of interest, were compared using a two-sided t-test and χ 2 test for continuous and categorical variables, respectively. Kaplan-Meier analysis and Cox proportional hazards regression was used to measure the association between the above exposures and all-cause and infection-related mortality in separate models. Detailed statistical methods are described in the supplementary document (section I). The association between the above exposures and sputum-smear and culture positivity at 2 months were analyzed using univariable and multivariable logistic regression. Potential confounders for multivariable analyses were identified by literature review and by exploratory univariable data analysis at P < 0.05 significance, depending on the assessed exposure.
The association of pre-existing CAD and ATS, and serum inflammatory markers, namely CRP, WBC, and NL ratio, was analyzed using univariable and multivariable linear regression analyses. We constructed a causal directed acyclic graph (cDAG) to represent our proposed mediation hypothesis linking the exposure (pre-existing CAD + AMI − or CAD + AMI + or ATS) and the outcome (all-cause and infection-related mortality during TB treatment) by using inflammatory markers (measured by CRP, NL ratio, WBC count) as the potential mediators.
Mortality. There was a higher all-cause mortality in the CAD + AMI + group (39/105 patients, 37.1%) and CAD + AMI − group (50/169 patients, 29.6%) compared to the non-CAD group (455/2393 patients, 19.0%) (P < 0.001) by χ 2 test ( Table 1). The log-rank test of the Kaplan-Meier analysis showed that patients in the CAD + AMI − and CAD + AMI + groups had significantly shorter survival compared to patients without CAD ( Fig. 2A; P < 0.001). After adjusting for age, gender, BMI, DM, HTN, cancer, CKD, asthma, COPD, liver cirrhosis, transplant status, sputum AFB status and cavitary disease at baseline, metformin use, statin use, calcium channel blocker use, patients in the CAD + AMI − group had a HR of 1.31 (95%CI 0.91-1.89) and the CAD + AMI + group had a HR of 2.04 (95%CI 1.38-3.00) for all-cause mortality ( Table 2). Patients with CAD + AMI − and CAD + AMI + had earlier infection-related mortality by the log-rank test of the Kaplan-Meier analysis ( Fig. 2B;

Association of ASCVD with systemic inflammation and its mediation of mortality.
After adjusting for confounders, the CAD + AMI + group had a significantly higher mean CRP (Regression co-efficient B (SE), 1.24 (0.62), P = 0.040) and the CAD + AMI − group had a non-significantly higher CRP (B (SE), 1.04(0.60), P = 0.087) compared to the non-CAD group in the linear regression analysis ( Table 3). The ATS group had a nonsignificantly higher CRP (B (SE), 0.49(0.68), P = 0.466) compared to the non-ATS group at baseline. No similar association was noted between the presence of CAD or ATS and other inflammatory markers, such as WBC or NL ratio at baseline. The causal directed acyclic graph (cDAG) representing our proposed mediation hypothesis linking the exposure (pre-existing ASCVD) and the outcome (all-cause and infection-related mortality during TB treatment) by using inflammatory markers (measured by CRP, NL ratio, WBC count) as the potential mediators is shown in Fig. 3. CRP mediated 66% of the association of CAD + AMI − with all-cause mortality (P = 0.048) and 73% of the association with infection-related mortality (P = 0.035) ( Table 4). CRP mediated 25% of the association of CAD + AMI + with all-cause mortality (P = 0.033) and 36% of the association with infection-related mortality (P = 0.042). CRP did not significantly mediate the association of ATS with all-cause or infection-related mortality ( Table 4). WBC and NL ratio were not found to mediate the association of ASCVD and mortality.

Stratification based on statin use among patients with ASCVD (CAD or ATS). Patient charac-
teristics. Among 355 patients with pre-existing ASCVD, 65 patients (18.3%) received statins according to the intention to treat definition and 45 patients (12.7%) received statins according to the per-protocol definition. Patient characteristics stratified by statin use according to the intention-to-treat exposure are shown in Table 5.
Mortality. Patients receiving statins (as per the intention-to treat definition) had a HR of 0.41 (95%CI, 0.19-0.84) for all-cause mortality and a HR of 0.42 (95%CI, 0.17-1.06) for infection-related mortality after adjusting for confounders (Table 6, Fig. 4A,B). Using the per-protocol definition, patients receiving statins had a HR of 0.41 (95%CI, 0.18-0.96) for all-cause mortality and a HR of 0.43 (95%CI, 0.15-1.25) for infection-related mortality after adjusting for confounders (Table 6). Sensitivity analyses excluding patients who died less than 30 days from treatment onset (Supplementary Table 1) and excluding patients who did not receive the required dose of statin from the comparison group (Supplementary Table 2) showed lower hazard for all-cause mortality but not infection-related mortality among statin users in the intention-to-treat analysis.

Discussion
Our retrospective cohort study found that a history of prior AMI and ATS was associated with higher all-cause and infection-related mortality during the first nine months after TB treatment initiation independent of confounding factors despite the comparison group having higher rates of cavitary TB disease at baseline. Pre-existing CAD (AMI − or AMI + ) or ATS was not associated with microbiological outcomes, namely sputum culture conversion or sputum smear AFB positivity at two months of TB treatment. The CAD + AMI − and CAD + AMI + groups had significantly higher levels of systemic inflammation at TB diagnosis, manifested by higher CRP levels in the univariable analysis. The multivariable analysis showed significant association only for the CAD + AMI + group, suggesting a relationship between the progression of CAD with systemic levels of inflammation. Higher NL ratios were noted in the ATS group only in univariable, but not multivariable, analysis.  36 . An important relationship has been demonstrated in the pathogenesis of TB and ASCVD 37 . The key hallmarks of atherosclerosis are LDL oxidation, foam cell formation and inflammation 38 . Preclinical studies have shown that Mtb infection of guinea pigs resulted in systemic oxidative stress, depletion of serum total antioxidant capacity and accumulation of oxidized LDL in granulomas 39 , with an increased expression of key atherosclerosis markers, such as scavenger receptor CD36 and lectin oxidized LDL receptor 1 39 .
A systematic review indicated that patients with TB have an increased risk of CAD 4 . Also, pulmonary TB was associated with a higher hazard of progression to AMI in a propensity score-matched analysis from a US insurance claims database 7 . Likewise, patients with TB have an increased risk of ischemic 9,10 , but not hemorrhagic stroke in the first three years after TB diagnosis 10 when compared to non-infected controls. Multiples cases of TB causing coronary arteritis and subsequent AMI have been reported 40,41 . Preclinical studies have shown that mycobacterial infection aggravates atherosclerosis formation in the aortas of hyperlipidemic, lipoprotein receptor CAD + AMI − , CAD + AMI + and ATS were assessed in separate models. Mediator: Inflammatory markers: CRP, WBC and NL ratio were assessed in separate models Outcomes: All-cause and infection related mortality were assessed in separate models. Confounders: Age, gender, body mass index, diabetes mellitus, hypertension, cancer, chronic kidney disease (stage 3-5), asthma, chronic obstructive pulmonary disease, liver cirrhosis, transplant status, baseline sputum acid fast bacilli status, cavitary disease at baseline, metformin use, statin use, and calcium channel blocker use. ATS, pre-existing atherothrombotic stroke; CAD, pre-existing coronary artery disease; CAD + AMI − , Pre-existing coronary artery disease without prior acute myocardial infarction; CAD + AMI + , Pre-existing coronary artery disease with prior acute myocardial infarction. CRP: C-reactive protein; NL ratio: Neutrophil lymphocyte ratio; WBC: Total leukocyte count (× 10 3 /µL). Table 3. Association of CAD and acute myocardial infarction (AMI) with inflammatory parameters. ATS: Atherothrombotic stroke, CAD + AMI + : Coronary artery disease with history of Acute myocardial infarction; CAD + AMI-: Coronary artery disease without history of Acute myocardial infarction, B: Regression co-efficient, SE: Standard error. # Adjusted for age, gender, body mass index (BMI), diabetes mellitus (DM), hypertension (HTN), cancer, chronic kidney disease (stage 3-5), asthma, chronic obstructive pulmonary disease (COPD), liver cirrhosis, transplant status, baseline sputum AFB status, cavitary disease at baseline, metformin use, statin use, and calcium channel blocker use. & Additionally adjusted for ATS. $ Additionally adjusted for CAD.  42 . These data suggest that M. tuberculosis and associated systemic inflammation may play a causative role in atherosclerosis. Prior reports in non-TB populations have shown that higher levels of systemic inflammation increase plaque instability, resulting in plaque rupture, fissuring, or erosion 14,[43][44][45][46] . In particular, patients with CAD and elevated CRP levels have higher rates of death and progression to AMI, relative to patients with lower CRP 47,48 . Our study demonstrated the consistency of these findings in ASCVD patients with TB as well, with CAD patients (AMI − or AMI + ) having greater levels of baseline CRP compared to patients without CAD. Similarly, patients with a history of ATS were found to have higher NL ratios than patients without ATS. Higher levels of inflammatory markers, such as CRP, WBC, and NL ratio, have been associated with a poor prognosis in patients with TB 8,18 . Our mediation analysis assessed whether the higher mortality observed in the CAD and ATS groups was mediated by systemic inflammation. We found that more than nearly one-quarter of the hazard of mortality in TB patients with AMI is mediated by systemic inflammation, as reflected by elevated serum CRP levels. These findings raise the intriguing possibility of targeting systemic inflammation as a potential intervention to reduce mortality and improve outcomes among TB patients with ASCVD.

P-value
Statins are commonly used agents used to reduce serum lipids and systemic inflammation to improve outcomes in patients with a history of or at risk for ASCVD. Prior systematic reviews have shown that statin use reduces TB incidence, both in patients with DM and in the general population 28,29 . Preclinical studies in macrophage and mouse models have demonstrated that statins enhance autophagy and phagosome maturation 24 , increase the proportion of NK cells, and increase secretion of the pro-inflammatory cytokines IL-1β and IL-12p70, thereby reducing the lung bacillary burden in M. tuberculosis-infected mice 24,49 . We found that the antitubercular activity of statins represents a class effect 50 , which is mediated by inhibition of cholesterol biosynthesis and autophagy via the AMPK-mTORC1-TFEB axis in macrophages 25 . These studies demonstrate that statins enhance the anti-tubercular activity of the first-line regimen and activate the host immune response against M. tuberculosis. Although a cohort study from an insurance claims database showed that TB treatment completion rates did not improve following statin therapy 30 , the study did not appropriately adjust for confounding factors. Furthermore, no previous clinical study has evaluated the effect of statin use on important TB-related outcomes, such as long-term lung function or sputum microbiological outcomes 51 . In our retrospective cohort, among patients with ASCVD, statin users had a lower hazard of all-cause mortality. We did not note a similar significant association between statin use and infection-related mortality. Though our cohort included only drug-susceptible TB patients, we believe that statin therapy may have similar beneficial effects in drug-resistant TB as well.
With respect to the strengths of our study, our large sample size enabled adjusting for multiple confounders. Availability of data on the serum inflammatory markers in a relatively large number of patients at baseline www.nature.com/scientificreports/ enabled mediation analysis to assess the effect of CAD mediated by systemic inflammation. Our study also has a few limitations. Follow-up levels of serum inflammatory markers following initiation of TB treatment would have enabled a trend of these parameters in patients with or without systemic inflammation at baseline. We did not have data on aspirin use, which can act as a confounding factor. We did not have a control population with respiratory or non-respiratory infections other than TB in our cohort to further assess the role played by systemic inflammation in cardiovascular diseases. The relatively small sample size of patients receiving statin therapy prevented an analysis of the effect of statin use on sputum smear and culture results.
In summary, patients with CAD and ATS have higher hazards of all-cause and infection-related mortality during the first nine months after TB treatment initiation. Elevated serum inflammation markers (CRP) mediate nearly one-quarter to one-third of this association when adjusted for confounders. However, pre-existing CAD or ATS was not associated with a difference in sputum culture or smear positivity at two months. Statin use was associated with lower all-cause mortality among patients with ASCVD. Randomized controlled trials are required to assess the utility of adjunctive statin therapy on microbiological and clinical outcomes in TB patients with and without ASCVD.