John F. Carlquist

Randomized Trial of Genotype-Guided Versus Standard Warfarin Dosing in Patients Initiating Oral Anticoagulation

Background— Pharmacogenetic-guided dosing of warfarin is a promising application of “personalized medicine” but has not been adequately tested in randomized trials. Methods and Results— Consenting patients (n=206) being initiated on warfarin were randomized to pharmacogenetic-guided or standard dosing. Buccal swab DNA was genotyped for CYP2C9 *2 and CYP2C9 *3 and VKORC1C1173T with a rapid assay. Standard dosing followed an empirical protocol, whereas pharmacogenetic-guided dosing followed a regression equation including the 3 genetic variants and age, sex, and weight. Prothrombin time international normalized ratio (INR) was measured routinely on days 0, 3, 5, 8, 21, 60, and 90. A research pharmacist unblinded to treatment strategy managed dose adjustments. Patients were followed up for up to 3 months. Pharmacogenetic-guided predicted doses more accurately approximated stable doses (P<0.001), resulting in smaller (P=0.002) and fewer (P=0.03) dosing changes and INRs (P=0.06). However, percent out-of-range INRs (pharmacogenetic=30.7%, standard=33.1%), the primary end point, did not differ significantly between arms. Despite this, when restricted to wild-type patients (who required larger doses; P=0.001) and multiple variant carriers (who required smaller doses; P<0.001) in exploratory analyses, results (pharmacogenetic=29%, standard=39%) achieved nominal significance (P=0.03). Multiple variant allele carriers were at increased risk of an INR of ≥4 (P=0.03). Conclusions— An algorithm guided by pharmacogenetic and clinical factors improved the accuracy and efficiency of warfarin dose initiation. Despite this, the primary end point of a reduction in out-of-range INRs was not achieved. In subset analyses, pharmacogenetic guidance showed promise for wild-type and multiple variant genotypes.

Infection With Chlamydia pneumoniae Accelerates the Development of Atherosclerosis and Treatment With Azithromycin Prevents It in a Rabbit Model

BACKGROUND Chlamydia pneumoniae infection has been associated with atherosclerosis by serological studies and detection of bacterial antigen within plaque. We sought to evaluate a possible causal role in an animal model. METHODS AND RESULTS Thirty New Zealand White rabbits were given three separate intranasal inoculations of either C pneumoniae (n = 20) or saline (n = 10) at 3-week intervals and fed chow enriched with a small amount (0.25%) of cholesterol. Immediately after the final inoculation, infected and control rabbits were randomized and begun on a 7-week course of azithromycin or no therapy. Three months after the final inoculation, rabbits were euthanatized and sections of thoracic aortas were blindly evaluated microscopically for maximal intimal thickness (MIT), percentage of luminal circumference involved (PLCI), and plaque area index (PAI) of atherosclerosis. Vascular chlamydial antigen was assessed by direct immunofluorescence. MIT differed among treatment groups (P=.009), showing an increase in infected rabbits (0.55 mm; SE = 0.15 mm) compared with uninfected controls (0.16 mm; SE = 0.06 mm) and with infected rabbits receiving antibiotics (0.20 mm; SE = 0.03 mm) (both P<.025), whereas MIT in infected/treated versus control rabbits did not differ. PLCI also tended to differ (P<.1) and PAI differed significantly (P<.01) among groups with a similar pattern. Chlamydial antigen was detected in 2 untreated, 3 treated, and 0 control animals. CONCLUSIONS Intranasal C pneumoniae infection accelerates intimal thickening in rabbits given a modestly cholesterol-enhanced diet. In addition, weekly treatment with azithromycin after infectious exposure prevents accelerated intimal thickening. These findings strengthen the etiologic link between C pneumoniae and atherosclerosis and should stimulate additional animal and human studies, including clinical antibiotic trials.

Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease.

OBJECTIVES The objectives of this study were to test prospectively for an association between Chlamydia and atherosclerosis by comparing the incidence of the pathogen found within atherosclerotic plaques in patients undergoing directional coronary atherectomy with a variety of control specimens and comparing the clinical features between the groups. BACKGROUND Previous work has suggested an association between Chlamydia pneumoniae infection and coronary atherosclerosis, based on the demonstration of increased serologic titers and the detection of bacteria within atherosclerotic tissue, but this association has not yet been regarded as established. METHODS Coronary specimens from 90 symptomatic patients undergoing coronary atherectomy were tested for the presence of Chlamydia species using direct immunofluorescence. Control specimens from 24 subjects without atherosclerosis (12 normal coronary specimens and 12 coronary specimens from cardiac transplant recipients with subsequent transplant-induced coronary disease) were also examined. RESULTS Coronary atherectomy specimens were definitely positive in 66 (73%) and equivocally positive in 5 (6%), resulting in 79% of specimens showing evidence for the presence of Chlamydia species within the atherosclerotic tissue. In contrast, only 1 (4%) of 24 nonatherosclerotic coronary specimens showed any evidence of Chlamydia. The statistical significance of this difference is a p value < 0.001. Transmission electron microscopy was used to confirm the presence of appropriate organisms in three of five positive specimens. No clinical factors except the presence of a primary nonrestenotic lesion (odds ratio 3.0, p = 0.057) predicted the presence of Chlamydia. CONCLUSIONS This high incidence of Chlamydia only in coronary arteries diseased by atherosclerosis suggests an etiologic role for Chlamydia infection in the development of coronary atherosclerosis that should be further studied.

Relation of vitamin D deficiency to cardiovascular risk factors, disease status, and incident events in a general healthcare population.

Vitamin D recently has been proposed to play an important role in a broad range of organ functions, including cardiovascular (CV) health; however, the CV evidence-base is limited. We prospectively analyzed a large electronic medical records database to determine the prevalence of vitamin D deficiency and the relation of vitamin D levels to prevalent and incident CV risk factors and diseases, including mortality. The database contained 41,504 patient records with at least one measured vitamin D level. The prevalence of vitamin D deficiency (≤30 ng/ml) was 63.6%, with only minor differences by gender or age. Vitamin D deficiency was associated with highly significant (p <0.0001) increases in the prevalence of diabetes, hypertension, hyperlipidemia, and peripheral vascular disease. Also, those without risk factors but with severe deficiency had an increased likelihood of developing diabetes, hypertension, and hyperlipidemia. The vitamin D levels were also highly associated with coronary artery disease, myocardial infarction, heart failure, and stroke (all p <0.0001), as well as with incident death, heart failure, coronary artery disease/myocardial infarction (all p <0.0001), stroke (p = 0.003), and their composite (p <0.0001). In conclusion, we have confirmed a high prevalence of vitamin D deficiency in the general healthcare population and an association between vitamin D levels and prevalent and incident CV risk factors and outcomes. These observations lend strong support to the hypothesis that vitamin D might play a primary role in CV risk factors and disease. Given the ease of vitamin D measurement and replacement, prospective studies of vitamin D supplementation to prevent and treat CV disease are urgently needed.

Randomized Secondary Prevention Trial of Azithromycin in Patients With Coronary Artery Disease and Serological Evidence for Chlamydia pneumoniae Infection The Azithromycin in Coronary Artery Disease: Elimination of Myocardial Infection with Chlamydia (ACADEMIC) Study

BACKGROUND Chlamydia pneumoniae commonly causes respiratory infection, is vasotropic, causes atherosclerosis in animal models, and has been found in human atheromas. Whether it plays a causal role in clinical coronary artery disease (CAD) and is amenable to antibiotic therapy is uncertain. METHODS AND RESULTS CAD patients (n=302) who had a seropositive reaction to C pneumoniae (IgG titers >/=1:16) were randomized to receive placebo or azithromycin, 500 mg/d for 3 days, then 500 mg/wk for 3 months. Circulating markers of inflammation (C-reactive protein [CRP], interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF]-alpha), C pneumoniae antibody titers, and cardiovascular events were assessed at 3 and 6 months. Treatment groups were balanced, with age averaging 64 (SD=10) years; 89% of the patients were male. Azithromycin reduced a global rank sum score of the 4 inflammatory markers at 6 (but not 3) months (P=0. 011) as well as the mean global rank sum change score: 531 (SD=201) for active drug and 587 (SD=190) for placebo (P=0.027). Specifically, change-score ranks were significantly lower for CRP (P=0.011) and IL-6 (P=0.043). Antibody titers were unchanged, and number of clinical cardiovascular events at 6 months did not differ by therapy (9 for active drug, 7 for placebo). Azithromycin decreased infections requiring antibiotics (1 versus 12 at 3 months, P=0.002) but caused more mild, primarily gastrointestinal, adverse effects (36 versus 17, P=0.003). CONCLUSIONS In CAD patients positive for C pneumoniae antibodies, global tests of 4 markers of inflammation improved at 6 months with azithromycin. However, unlike another smaller study, no differences in antibody titers and clinical events were observed. Longer-term and larger studies of antichlamydial therapy are indicated.

Evaluation of C-reactive protein, an inflammatory marker, and infectious serology as risk factors for coronary artery disease and myocardial infarction☆

OBJECTIVES We sought to test whether C-reactive protein (CRP) and seropositivity to any of three infectious agents are associated with angiographic coronary artery disease (CAD) and clinical myocardial infarction (MI). BACKGROUND CRP, an inflammatory marker, is reported to predict risk of MI. The stimulus for CRP is unknown but might include infection. Chlamydia pneumoniae, cytomegalovirus and Helicobacter pylori have been linked to risk of MI or CAD. METHODS Blood samples were collected from 363 patients undergoing coronary arteriography and tested for CRP and IgG titers to the infectious agents. RESULTS CRP was higher in patients with CAD (1.32 mg/dl [SE 0.22, n = 80] vs. 0.58 mg/dl [SE 0.11 mg/dl, n = 109], p = 0.004) and in those with MI (2.05 mg/dl [SE 0.36, n = 47] vs. 0.54 mg/dl [SE 0.08, n = 133], p = 0.0002) than in respective control subjects. Seropositivity for each agent was present in a high proportion of patients with CAD (58% to 77%) or MI (54% to 75%) as well as in control subjects (no CAD: 46% to 74%; no MI: 50% to 77%). However, subjects seropositive to both C. pneumoniae and H. pylori had an increased prevalence of CAD (odds ratio [OR] 2.6, p = 0.02) and MI (OR 2.0, p = 0.15) and tended to have higher CRP levels (1.07 mg/dl [SE 0.16]) than those seronegative to both infectious agents (0.53 mg/dl [SE 0.10], p = 0.06). CONCLUSIONS CRP is elevated in patients with CAD (more than twofold) and in those with MI (fourfold). Infectious serology is highly prevalent in both patients and control subjects. Seropositivity to both C. pneumoniae and H. pylori (but not one agent alone) may predict increased risk and may be associated with higher CRP levels. Infectious serology may be less predictive than previously suggested, but the cause of inflammation in CAD and MI deserves further study.

Plasma Homocysteine Predicts Mortality Independently of Traditional Risk Factors and C-Reactive Protein in Patients With Angiographically Defined Coronary Artery Disease

BackgroundPlasma homocysteine (tHCY) has been associated with coronary artery disease (CAD). We tested whether tHCY also increases secondary risk, after initial CAD diagnosis, and whether it is independent of traditional risk factors, C-reactive protein (CRP), and methylenetetrahydrofolate reductase (MTHFR) genotype. Methods and ResultsBlood samples were collected from 1412 patients with severe angiographically defined CAD (stenosis ≥70%). Plasma tHCY was measured by fluorescence polarization immunoassay. The study cohort was evaluated for survival after a mean of 3.0±1.0 years of follow-up (minimum 1.5 years, maximum 5.0 years). The average age of the patients was 65±11 years, 77% were males, and 166 died during follow-up. Mortality was greater in patients with tHCY in tertile 3 than in tertiles 1 and 2 (mortality 15.7% versus 9.6%, P =0.001 [log-rank test], hazard ratio [HR] 1.63). The relative hazard increased 16% for each 5-&mgr;mol/L increase in tHCY (P <0.001). In multivariate Cox regression analysis, controlling for univariate clinical and laboratory predictors, elevated tHCY remained predictive of mortality (HR 1.64, P =0.009), together with age (HR 1.72 per 10-year increment, P <0.0001), ejection fraction (HR 0.84 per 10% increment, P =0.0001), diabetes (HR 1.98, P =0.001), CRP (HR 1.42 per tertile, P =0.004), and hyperlipidemia. Homozygosity for the MTHFR variant was weakly predictive of tHCY levels but not mortality. ConclusionsIn patients with angiographically defined CAD, tHCY is a significant predictor of mortality, independent of traditional risk factors, CRP, and MTHFR genotype. These findings increase interest in tHCY as a secondary risk marker and in secondary prevention trials (ie, with folate/B vitamins) to determine whether reduction in tHCY will reduce risk.

Randomized Secondary Prevention Trial of Azithromycin in Patients With Coronary Artery Disease Primary Clinical Results of the ACADEMIC Study

BackgroundChlamydia pneumoniae is associated with coronary artery disease (CAD), although its causal role is uncertain. A small preliminary study reported a >50% reduction in ischemic events by azithromycin, an antibiotic effective against C pneumoniae, in seropositive CAD patients. We tested this prospectively in a larger, randomized, double-blind study. Methods and ResultsCAD patients (n=302) seropositive to C pneumoniae (IgG titers ≥1:16) were randomized to placebo or azithromycin 500 mg/d for 3 days and then 500 mg/wk for 3 months. The primary clinical end point included cardiovascular death, resuscitated cardiac arrest, nonfatal myocardial infarction (MI), stroke, unstable angina, and unplanned coronary revascularization at 2 years. Treatment groups were balanced, and azithromycin was generally well tolerated. During the trial, 47 first primary events occurred (cardiovascular death, 9; resuscitated cardiac arrest, 1; MI, 11; stroke, 3; unstable angina, 4; and unplanned coronary revascularization, 19), with 22 events in the azithromycin group and 25 in the placebo group. There was no significant difference in the 1 primary end point between the 2 groups (hazard ratio for azithromycin, 0.89; 95% CI, 0.51 to 1.61;P =0.74). Events included 9 versus 7 occurring within 6 months and 13 versus 18 between 6 and 24 months in the azithromycin and placebo groups, respectively. ConclusionsThis study suggests that antibiotic therapy with azithromycin is not associated with marked early reductions (≥50%) in ischemic events as suggested by an initial published report. However, a clinically worthwhile benefit (ie, 20% to 30%) is still possible, although it may be delayed. Larger (several thousand patient), longer-term (≥3 to 5 years) antibiotic studies are therefore indicated.

A Randomized and Clinical Effectiveness Trial Comparing Two Pharmacogenetic Algorithms and Standard Care for Individualizing Warfarin Dosing (CoumaGen-II)

Background— Warfarin is characterized by marked variations in individual dose requirements and a narrow therapeutic window. Pharmacogenetics (PG) could improve dosing efficiency and safety, but clinical trials evidence is meager. Methods and Results— A Randomized and Clinical Effectiveness Trial Comparing Two Pharmacogenetic Algorithms and Standard Care for Individualizing Warfarin Dosing (CoumaGen-II) comprised 2 comparisons: (1) a blinded, randomized comparison of a modified 1-step (PG-1) with a 3-step algorithm (PG-2) (N=504), and (2) a clinical effectiveness comparison of PG guidance with use of either algorithm with standard dosing in a parallel control group (N=1866). A rapid method provided same-day CYP2C9 and VKORC1 genotyping. Primary outcomes were percentage of out-of-range international normalized ratios at 1 and 3 months and percentage of time in therapeutic range. Primary analysis was modified intention to treat. In the randomized comparison, PG-2 was noninferior but not superior to PG-1 for percentage of out-of-range international normalized ratios at 1 month and 3 months and for percentage of time in therapeutic range at 3 months. However, the combined PG cohort was superior to the parallel controls (percentage of out-of-range international normalized ratios 31% versus 42% at 1 month; 30% versus 42% at 3 months; percentage of time in therapeutic range 69% versus 58%, 71% versus 59%, respectively, all P<0.001). Differences persisted after adjustment for age, sex, and clinical indication. There were fewer percentage international normalized ratios ≥4 and ⩽1.5 and serious adverse events at 3 months (4.5% versus 9.4% of patients, P<0.001) with PG guidance. Conclusions— These findings suggest that PG dosing should be considered for broader clinical application, a proposal that is being tested further in 3 major randomized trials. The simpler 1-step PG algorithm provided equivalent results and may be preferable for clinical application. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00927862.

Integration of genetic, clinical, and INR data to refine warfarin dosing

Well‐characterized genes that affect warfarin metabolism (cytochrome P450 (CYP) 2C9) and sensitivity (vitamin K epoxide reductase complex 1 (VKORC1)) explain one‐third of the variability in therapeutic dose before the international normalized ratio (INR) is measured. To determine genotypic relevance after INR becomes available, we derived clinical and pharmacogenetic refinement algorithms on the basis of INR values (on day 4 or 5 of therapy), clinical factors, and genotype. After adjusting for INR, CYP2C9 and VKORC1 genotypes remained significant predictors (P < 0.001) of warfarin dose. The clinical algorithm had an R2 of 48% (median absolute error (MAE): 7.0 mg/week) and the pharmacogenetic algorithm had an R2 of 63% (MAE: 5.5 mg/week) in the derivation set (N = 969). In independent validation sets, the R2 was 26–43% with the clinical algorithm and 42–58% when genotype was added (P = 0.002). After several days of therapy, a pharmacogenetic algorithm estimates the therapeutic warfarin dose more accurately than one using clinical factors and INR response alone.