Abstract
Background and aims
Ischemic heart disease (IHD) accounts for one-third of annual deaths in the U.S. and mortality rates vary by ethnicity. The association between adherence to dietary guidelines for fruit and vegetable intake with IHD mortality among different ethnic groups has not previously been examined.
Methods and results
A prospective cohort design was used to examine the incidence of fatal IHD among participants in the Multiethnic Cohort Study. Participants included 164,617 men and women from five ethnic groups: African American, Native Hawaiian, Japanese American, Latino, and Caucasian. Cox proportional hazards models, stratified by ethnicity and sex, were used to examine associations between adherence with recommended dietary guidelines for fruit and vegetable intake and risk for fatal IHD. The results did not provide evidence that the association between adherence with dietary recommendations for fruit or vegetable intake and IHD mortality varies by ethnicity. Pooled data did provide evidence that adhering to the recommendations for vegetables lowered risk among men (RR Z 0.84, 95% CI: 0.74e0.96) and women (RR Z 0.80, 95% CI: 0.69e0.94). No significant effects were observed for fruit intake.
Conclusions
The effect of dietary intake of fruit and vegetables did not vary by ethnicity, providing evidence that recommendations do not need to be individualized for these special populations. The protective effect observed for vegetable intake among both sexes confirms previous findings and supports the evidence base for promoting diet modification in this direction.
Keywords: Dietary recommendations, fatal ischemic heart disease, ethnicity, Multiethnic Cohort Study
INTRODUCTION
Ischemic heart disease (IHD) is the most common type of heart disease with the highest mortality rate in the U.S. It accounts for almost one-third of annual deaths (approximately 425,000 deaths in 2006) and is also projected to be a major cause of death in the developing world [1] and [2]. The rate of fatal IHD varies substantially by ethnic group within the U.S. The age-adjusted death rate of IHD, per 100,000 US population, was 161.6 in African Americans, 136.0 in non-Hispanic Caucasians, 106.4 in Latinos, 97.4 in American Indians or Alaska Natives, and 77.1 in Asian or Pacific Islanders in 2005 [2].
Environmental factors, especially diet, have long been suspected to cause these ethnic variations but few large-scale datasets have been available. There is evidence of a protective effect of fruit and vegetables on risk of IHD but most previous studies have not used standardized methodologies or been conducted in large multiethnic populations [3], [4] and [5]. Previous studies in the Multiethnic Cohort (MEC) have revealed ethnic differences in the types of food consumed, as well as variation in adherence with dietary recommendations for fruit and vegetable intake [6] and [7]. The dietary recommendations developed by the United States Department of Agriculture (USDA) for daily consumption of fruits and vegetables are standardized for the general U.S. population [8]. However, to our knowledge, there are no studies examining the association of adherence to these recommendations with risk of fatal IHD among different ethnic groups.
Since the percentage of the U.S. population from minority ethnic backgrounds is increasing, there is a recognized need for ethnic-specific health data to identify high risk subgroups [9]. The aim of this study was to examine associations with adherence to the USDA dietary recommendations for dietary intake of fruits and vegetables and risk of fatal IHD among a large representative sample of five ethnic groups in the U.S.
METHODS
Study population
Recruitment procedures, study design and baseline characteristics have been reported previously [10]. In brief, MEC participants included representative samples of African–American, Japanese–American, Native Hawaiian, Latino, and Caucasian men and women between the ages of 45 and 75, living in Hawaii and the Los Angeles County area between 1993 and 1996. All participants completed a comprehensive mailed questionnaire at baseline including a detailed quantitative food frequency questionnaire (QFFQ). A total of 201,257 respondents from the five ethnic groups completed the questionnaire, with response rates varying from 20% among Latinos to 49% in Japanese–Americans. Participants with missing smoking information (n = 6080), implausible diets based on energy and macronutrient intakes as well as food group consumption (n = 12,346), implausible or missing anthropometric information (n = 3251), and who reported a history of heart attack or angina (n = 14,880), were excluded, leaving a total of 72,866 men and 91,751 women in the present analyses. All participants provided informed consent, and the study protocol was approved by the institutional review boards of the University of Hawaii and the University of Southern California.
Dietary assessment
The development of the QFFQ has been described elsewhere (11). In brief, three-day measured dietary records from 60 men and 60 women of each ethnic group served as the basis for the selection of food items for the QFFQ. A minimum set of food items contributing at least 85% to the consumption of nutrients of interest for each ethnic group were selected, complemented by ethnic-specific foods, irrespective of their nutrient contribution. The QFFQ captured intake frequency over the past year, based on 8–9 categories ranging from “Never or hardly ever” to “2 or more times a day”, as well as the amount of food consumed, based on a choice of three portion sizes represented in both photographs and amounts.
A validation and calibration substudy using 24-hour dietary recalls was conducted (12). Average correlation coefficients for all nutrients ranged from 0.26 in African-American women to 0.57 in Caucasian men. Average correlations for nutrient densities ranged from 0.57 to 0.74 across sex- and ethnic-specific strata.
A food composition table (FCT) specific for this multiethnic population was developed at the University of Hawaii Cancer Center [10]. The FCT includes a large recipe database and many unique foods consumed by the multiethnic population. The food groups and servings were based on the USDA dietary guidelines, including vegetables, fruit, meat and meat alternatives, grains and dairy products [8]. The number of servings across the appropriate food items on the QFFQ was summed to calculate each individual's number of food group servings. Mixed dishes were broken down into their component ingredients. Mean servings of food groups by ethnic-sex group were presented previously [6] and [7].
The USDA recommendations are based on age (≤50 years and >50 years) for dairy products and caloric intake for the other four food groups. Subjects were categorized into four groups according to their caloric intake: ≤6 698 kJ (≤1 600 kcal), 6 699 – 9 209 kJ (1 601 – 2 200 kcal), 9 210 – 11 721 kJ (2 201 – 2 800 kcal), >11 721 kJ (>2 800 kcal). The recommendation for these caloric intake groups were 3, 3, 4, and 5 servings/daily for vegetables, respectively; 2, 3, 3, and 4 servings/day for fruit, respectively; 2, 2, 2, 3 servings/day for meat, respectively; 6, 6, 9, and 11 servings/day for grains, respectively. For dairy products the USDA requirement are 2 servings/day for individuals ≤50 years and 3 servings/day for subjects >50 years. The percentage of the daily servings of each food group over its recommended servings at the matched age and caloric intake level was calculated to determine the degree of adherence.
Identification of heart disease deaths
For this analysis, we linked the MEC data with state death files and the National Death Index. Death from IHD includes ICD9 codes 410–414.9 or ICD10 codes I20–125.9. Follow-up for this study ended at the earliest of the following dates: the date of death or December 31st, 2001 (the closure data for this study).
Statistical analysis
To determine the associations between adherence to the USDA food group serving recommendations and fatal ischemic heart disease, we applied Cox proportional hazards models using age as the time metric to calculate relative risks (RR) and 95% confidence intervals (CIs). Analyses were conducted using a binary outcome measure for adherence with recommendations for dietary intake (<100% adherence versus ≥100%) Separate models for men and women are presented. The following covariates were used as adjustment factors: ethnicity when appropriate (as a stratum variable), time on study (≤2, 3–5, and >5 years, as a stratum variable), years of education, energy intake (logarithmically transformed), smoking (including current smoking, past smoking, and pack-years), body mass index, physical activity (defined as average hours of moderate or vigorous physical activity per day), history of diabetes, and alcohol intake (grams per day). In addition, the models for women included history of hormone replacement therapy. The results for dairy are presented with as well as without additional adjustment for saturated fat. After evaluating complex smoking models, including time-dependent duration of smoking and time since quitting, we found that a model with indicator variables for current and former smokers and pack-years was appropriate. Tests based on Schoenfeld residuals showed no evidence that any of our analyses violated proportional hazards assumptions. All analyses were performed using SAS software, version 9.1 (SAS Institute Inc., Cary, NC, 2005), and all p values were 2-sided.
RESULTS
A total of 1,140 male and 811 female cases of fatal IHD were identified in the MEC through December 31st, 2001. The total cohort and cases are described in Table 1. Cases reported more pack-years of cigarette smoking than the entire cohort. In addition, a higher proportion of cases was current smokers, reported a history of diabetes and hypertension, and had ≤10 years of education compared to all participants. In addition, the cases had a lower proportion of married persons, particularly among women.
Table 1.
Characteristics of cases of fatal ischemic heart disease and total participants
| Characteristics | Men | Women | ||
|---|---|---|---|---|
| Cases (n=1 140) |
Total participants (n=72 866) |
Cases (n=811) |
Total participants (n=91 751) |
|
| Age at cohort entry (years) | 65.8 ± 7.7 | 65.7 ± 7.6 | 66.4 ± 7.1 | 59.3 ± 8.8 |
| Energy intake (kcal) | 2 206 ± 1 028 | 2 309 ± 974 | 1 866 ± 941 | 1 885 ± 834 |
| % energy from fat | 31.6 ± 7.1 | 30.3 ± 7.1 | 30.5 ± 7.3 | 29.7 ± 7.0 |
| % energy from saturated fat | 9.6 ± 2.7 | 9.0 ± 2.6 | 9.1 ± 2.6 | 8.7 ± 2.6 |
| % energy from alcohol | 3.4 ± 7.5 | 4.2 ± 7.4 | 1.6 ± 5.8 | 1.6 ± 4.7 |
| Hours in moderate or vigorous activity per day | 1.0 ±1.3 | 1.3 ±1.5 | 0.8 ± 1.0 | 1.1 ± 1.2 |
| Pack-years (number of cigarettes per day × years smoked / 20) | 18.7 ± 19.0 | 13.7 ± 16.4 | 10.8 ± 15.2 | 6.5 ± 12.0 |
| Ethnicity (%) | ||||
| Caucasian | 23 | 26 | 19 | 26 |
| African American | 23 | 13 | 40 | 18 |
| Hawaiian | 9 | 7 | 8 | 7 |
| Japanese | 22 | 31 | 16 | 29 |
| Latino | 23 | 23 | 17 | 20 |
| BMI (kg/m2) (%) | ||||
| ≤ 18.5 | 2 | 24 | 5 | 4 |
| 18.5 −25.0 | 41 | 28 | 35 | 48 |
| 25.1 – 30.0 | 42 | 22 | 31 | 30 |
| > 30 | 15 | 26 | 28 | 18 |
| Smoking status (%) | ||||
| Never smoked | 24 | 32 | 45 | 57 |
| Past smoker | 50 | 50 | 30 | 29 |
| Current smoker | 26 | 18 | 25 | 14 |
| Repeatedly consumed alcohol (%) | 49 | 63 | 27 | 39 |
| Medical history (%) | ||||
| History of Diabetes | 30 | 11 | 37 | 10 |
| History of Hypertension | 58 | 38 | 69 | 36 |
| Education (%) | ||||
| Graduated college | 19 | 31 | 13 | 26 |
| Grade 11/12-some college | 56 | 53 | 61 | 58 |
| ≤10yrs education | 25 | 16 | 26 | 16 |
| Currently Married (%) | 71 | 77 | 41 | 60 |
Baseline levels of adherence for the participants included in these analyses are presented in Table 2. Across most ethnic groups, women exhibited higher levels of adherence than men. Across ethnic groups, adherence levels varied more for vegetable intake, ranging by approximately 13 and 10% for the highest level of vegetable intake (for women and men respectively). The proportion of participants whose fruit intake met or exceeded the dietary recommendations ranged among the different ethnic groups by approximately 6% for women and 8% for men.
Table 2.
Adherence to the USDA dietary recommendations and risk of death from ischemic heart disease among men and women by ethnic group1
| African American | Native Hawaiian | Japanese American | Latino | Caucasian | |
|---|---|---|---|---|---|
| MEN | |||||
| Cases/ Non-cases | 267 / 9 290 | 99 / 4 830 | 247 / 22 292 | 269 / 16 540 | 258 /18 774 |
| RR (95% CI) | RR (95% CI) | RR (95% CI) | RR (95% CI) | RR (95% CI) | |
| No | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Vegetables | 0.92 (0.70–1.21) | 1.25 (0.81–1.94) | 0.87 (0.66–1.14) | 0.70 (0.53–0.91) | 0.81 (0.62–1.05) |
| Fruits | 0.93 (0.71–1.20) | 1.07 (0.70–1.65) | 1.13 (0.86–1.47) | 0.85 (0.66–1.10) | 0.77 (0.59–1.01) |
| Meat | 1.56 (1.16–2.10) | 1.11 (0.69–1.77) | 1.04 (0.77–1.39) | 1.11 (0.83–1.49) | 1.20 (0.90–1.60) |
| Grains | 0.93 (0.70–1.24) | 0.92 (0.59–1.42) | 1.01 (0.75–1.36) | 1.01 (0.77–1.31) | 1.14 (0.88–1.49) |
| Dairy | 1.47 (0.88–2.45) | 0.77 (0.28–2.16) | 1.24 (0.45–3.37) | 1.30 (0.88–1.93) | 1.48 (1.01–2.17) |
| Dairy2 | 1.41 (0.84–2.35) | 0.69 (0.25–1.94) | 1.21 (0.44–3.28) | 1.24 (0.83–1.84) | 1.33 (0.90–1.96) |
| WOMEN | |||||
| Cases / Non-cases | 322 / 16 479 | 65 / 6 507 | 126 / 26 574 | 141 / 18 144 | 157 / 23 236 |
| RR (95% CI) | RR (95% CI) | RR (95% CI) | RR (95% CI) | RR (95% CI) | |
| No | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Vegetables | 0.88 (0.68–1.14) | 0.61 (0.33–1.09) | 0.72 (0.48–1.07) | 0.99 (0.67–1.47) | 0.70 (0.49–0.99) |
| Fruits | 0.88 (0.69–1.13) | 0.76 (0.44–1.33) | 1.11 (0.78–1.73) | 0.93 (0.64–1.35) | 1.04 (0.74–1.47) |
| Meat | 1.05 (0.79–1.38) | 0.75 (0.40–1.41) | 0.99 (0.62–1.58) | 1.77 (1.14–2.75) | 0.97 (0.62–1.51) |
| Grains | 1.35 (1.03–1.77) | 1.21 (0.69–2.14) | 0.73 (0.49–1.10) | 0.81 (0.54–1.23) | 1.13 (0.77–1.66) |
| Dairy | 0.98 (0.56–1.73) | 1.38 (0.52–3.68) | 1.37 (0.43–4.42) | 0.88 (0.46–1.69) | 0.79 (0.40–1.54) |
| Dairy 2 | 0.99 (0.56–1.73) | 1.41 (0.53–3.76) | 1.38 (0.43–4.44) | 0.92 (0.48–1.77) | 0.77 (0.39–1.51) |
Cox regression with age as the time metric adjusted for ethnicity, time on study, maximum years of education, energy intake (logarithmically transformed), smoking behavior (including current smoking, past smoking and pack-years), body mass index (kg/m2), physical activity (defined as average hours of moderate or vigorous physical activity per day), history of diabetes, and alcohol intake (grams per day);
Saturated fat intake was additionally adjusted for in this analysis of dairy product intake.
The ethnic-sex specific associations between degree of adherence to the food group recommendations and risk of fatal IHD are presented in Table 3. Significant protective associations were observed for vegetable intake among Latino men (RR = 0.62, 95% CI: 0.40–0.95) and Caucasian women (RR = 0.64, 95% CI: 0.43–0.96) consuming between 100 and149% of recommended intake levels and African American women with all intake levels except the third quantile (RR = 0.64, 95% CI: 0.43–0.96 for the highest quantile). Inconsistent results were observed for fruit intake. An elevated risk for ischemic heart disease mortality was observed among African American men with fruit intake in the second quintile (RR = 1.98, 95% CI: 1.38–2.83), while a protective association was observed among Caucasian men with fruit intake in the third quantile (RR = 0.64, 95% CI: 0.43–0.96) and no associations were observed among women. As comparison of the confidence intervals across strata did not provide any strong evidence that ethnicity modified the effect of adherence with recommended dietary intake on risk of IHD fatality, pooled results for men and women are presented in the final column of Table 3. A protective association was observed among men and women with vegetable intake at or above the dietary recommendation. A significant protective association was also observed among women with vegetable intake between 50 and 74% of the dietary recommendation (RR = 0.67, 95% CI: 0.50–0.88). A significant decreasing trend for the association between vegetable intake and ischemic heart disease was observed among Latino men (ptrend = 0.02) and men overall (ptrend = 0.05).
Table 3.
Adherence to the USDA dietary recommendations and risk of death from ischemic heart disease among men and women1
| Men | Women | |
|---|---|---|
| Cases / Non-cases | 1 140 / 71 726 | 811 / 90 940 |
| RR (95% CI) | RR (95% CI) | |
| No | 1.00 | 1.00 |
| Vegetables | 0.84 (0.74–0.96) | 0.80 (0.69–0.94) |
| Fruits | 0.91 (0.80–1.03) | 0.94 (0.80–1.09) |
| Meat | 1.20 (1.04–1.37) | 1.08 (0.91–1.30) |
| Grains | 1.01 (0.88–1.15) | 1.06 (0.90–1.25) |
| Dairy | 1.33 (1.06–1.66) | 0.91 (0.66–1.26) |
| Dairy 2 | 1.25 (0.99–1.57) | 0.91 (0.66–1.26) |
Cox regression with age as the time metric adjusted for ethnicity, time on study, maximum years of education, energy intake (logarithmically transformed), smoking behavior (including current smoking, past smoking and pack-years), body mass index (kg/m2), physical activity (defined as average hours of moderate or vigorous physical activity per day), history of diabetes, and alcohol intake (grams per day). The models for women were additionally adjusted for use of hormone replacement therapy.
Saturated fat intake was additionally adjusted for in this analysis of dairy product intake.
Ethnic-specific results for adherence to the USDA recommendation based on a binary outcome measure (<100% adherence versus ≥100%) are presented in Table 4. With the exception of Native Hawaiian men, the point estimates for adherence with vegetable intake suggested a protective effect for the risk of fatal IHD among all groups. However, in the ethnicity-stratified models, this effect was only significant among Latino men (RR = 0.70, 95% CI: 0.53–0.91) and Caucasian women (RR = 0.70, 95% CI: 0.49–0.99). No effects were apparent for associations with fruit intake and risk of IHD. Pooled results for men and women with the ethnic groups combined are presented in the final column of Table 4. Adherence with the recommendations for vegetable intake was associated with a significantly lower risk of IHD among both men (RR = 0.84, 95% CI: 0.74–0.96) and women (RR = 0.80, 95% CI: 0.69–0.94). The point estimates for the associations with fruit intake suggested a protective effect in both men and women, but these results did not reach statistical significance.
Table 4.
Associations between adherence with dietary recommendations and risk for ischemic heart disease mortality1
| African American |
Native Hawaiian |
Japanese American |
Latino | Caucasian | All ethnic groups |
|
|---|---|---|---|---|---|---|
| RR (95% CI) | RR (95% CI) |
RR (95% CI) | RR (95% CI) | RR (95% CI) | RR (95% CI) | |
| Men – cases/non- cases |
267/9290 | 99/4830 | 247/22,292 | 269/16,540 | 258/18,774 | 1140/71,726 |
| Referenceb | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Vegetables | 0.92 (0.70– 1.21) |
1.25 (0.81– 1.94) |
0.87 (0.66– 1.14) |
0.70 (0.53– 0.91) |
0.81 (0.62– 1.05) |
0.84 (0.74– 0.96) |
| Fruits | 0.93 (0.71– 1.20) |
1.07 (0.70– 1.65) |
1.13 (0.86– 1.47) |
0.85 (0.66– 1.10) |
0.77 (0.59– 1.01) |
0.91 (0.80– 1.03) |
| Women – cases/non- cases |
322/16,479 | 65/6507 | 126/26,574 | 141/18,144 | 157/23,236 | 811/90,940 |
| Reference2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Vegetables | 0.88 (0.68– 1.14) |
0.61 (0.33– 1.09) |
0.72 (0.48– 1.07) |
0.99 (0.67– 1.47) |
0.70 (0.49– 0.99) |
0.80 (0.69– 0.94) |
| Fruits | 0.88 (0.69– 1.13) |
0.76 (0.44– 1.33) |
1.11 (0.78– 1.73) |
0.93 (0.64– 1.35) |
1.04 (0.74– 1.47) |
0.94 (0.80– 1.09) |
Cox regression with age as the time metric adjusted for ethnicity, time on study, maximum years of education, energy intake (logarithmically transformed), smoking behavior (including current smoking, past smoking and pack-years), body mass index (kg/m2), physical activity (defined as average hours of moderate or vigorous physical activity per day), history of diabetes, alcohol intake (grams per day) and food group intake (meat, grain, dairy products). Statistically significant (p ≤ 0.05) values are indicated in bold.
Reference group: nonadherent with dietary recommendations.
Discussion
To our knowledge, this study is the first study to explore the effects of adherence to the USDA recommendations for the five food groups and risk of fatal IHD among multiple ethnic groups in the United States. As the proportion of ethnic minorities in the U.S. population increases, it is important to determine if risk factors for disease vary among ethnic groups in order to identify high risk groups and target appropriate interventions. The impact of very cost-effective interventions such as diet modification are particularly relevant considering the growing burden of chronic diseases such as IHD.
The results from this large multi-ethnic cohort suggest that diet does not differentially impact the risk of IHD among different ethnic groups. However, our results did show that, overall, men and women who met or exceeded the recommendations for vegetable intake had a reduced risk of CHD death. We also examined trends based on the level of adherence with recommendations for vegetable intake (data not shown) and found that the protective effect increased significantly with vegetable consumption. Similar findings on the protective effect of vegetable intake have been reported by others (13;14). This suggests there may be need to re-evaluate the dietary guidelines to reflect an even higher recommendation of vegetables in an effort to reduce risk of chronic disease and improve overall health, particularly in groups known to be at higher risk (e.g., people with family history) (15,16).
Some previous studies found an inverse association between risk of IHD and consumption of vegetables alone [17], as well as vegetables and fruit combined [3],[4] and [18]. A number of components of fruit and vegetables, such as polyunsaturated fatty acids, plant sterols, fiber, polyphenolic compounds, vitamin C, and vitamin E are considered as protective factors for IHD [19], [20] and [21]. Phytochemicals, including fiber, have the potential to reduce hyperlipidemia and the reabsorption of bile salts[22] and [23]. However, one cohort study found no association between dietary fiber and IHD mortality [15]. Vitamin E and vitamin C have been shown to be particularly beneficial in the prevention of inflammation in atherosclerotic plaque development, and in endothelial dysfunction, respectively [16] and [24]. In contrast, vitamin D has been shown to be a risk factor for IHD [19], and a meta-analysis of cohort studies concluded that the specific components in fruit and vegetables responsible for the protective effects are unknown [25]. It is also possible that the null findings for fruit consumption in our study are due to opposing effects of different types of fruit. More studies on subgroups of fruit are necessary to clarify the current observations.
The MEC study provided a unique opportunity to examine associations between diet, IHD mortality and ethnicity using large representative samples from five ethnic minorities living in the United States. Collection of dietary information using a common QFFQ, including ethnic-specific foods and portion sizes, facilitated comparison across ethnic groups, and with other studies. We also had access to information on a wide variety of covariates which allowed adjustment of our models for possible confounders. Limitations of this study include possible over reporting of food consumption, as well as recall bias [26]. However, the QFFQ used in the MEC has been validated and appears to capture total intake relatively well [11]. In addition, changes in diet over a person's lifetime, which cannot be captured with the assessment of food consumption over a narrow time period, may have influenced the associations between diet and risk for IHD. Although this analysis was based on large samples of each ethnic group, there were a large number of exclusions, and with the variation in response rate among the different ethnic groups, selection bias is also a concern. Nonetheless, our findings are consistent with previous reports, which support the validity of our findings.
Conclusions
The results from this study did not provide any evidence that the effects on the risk of IHD mortality associated with adherence to the U.S. dietary recommendations for fruit and vegetable intake vary by ethnicity. However, given the increasing ethnic diversity in the United States and the growing burden of chronic diseases such as IHD, it is particularly salient to examine the potential impact of simple cost-effective interventions such as diet modification. The protective effect of vegetables observed in this study is supported by previous work and further demonstrates the importance of promoting diet modification in this direction.
Acknowledgments
This research was funded by the American Heart Association of Hawaii (Beginning Grant-in-Aid, grant number 0265287Z). The study was also supported by the National Cancer Institute (grant number R37 CA54821) and the United States Department of Agriculture (USDA-NRI New Investigator Award, grant number 2002-00793). None of the sponsors had any role in the study design, analysis or interpretation of the data, or manuscript development.
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