Pediatric Pulmonology 50:441–446 (2015)
Association Between Serum 25-Hydroxyvitamin D Level and Pulmonary Exacerbations in Cystic Fibrosis Michelle B. Vanstone, MD,1 Marie E. Egan, MD,2 Jane H. Zhang, and Thomas O. Carpenter, MD1*
PhD,
3
Summary. Objective: To identify effects of vitamin D status, as defined by circulating 25hydroxyvitamin D (25-OHD) levels on the annual frequency of pulmonary exacerbations (Pex) and hospitalizations, on standard measures of pulmonary function, and to identify determinants of 25-OHD levels in pediatric patients with cystic fibrosis (CF). Hypothesis: Higher levels of serum 25-OHD would be associated with fewer Pex and hospitalizations, and improved lung function based on pulmonary function tests (PFTs). Study design: Retrospective chart review of 53 pediatric patients from January 2011 to December 2011. Significant relationships were examined using linear and logistic regression analyses. Patient Selection: Patients ages 5 through 22 years followed at the CF Care Center and Clinic at Yale-New Haven Hospital, New Haven, CT., who had at least four clinic visits and at least one 25-OHD measurement between January 1, 2011 and December 31, 2011. Results: Serum 25-OHD level and gender were strong independent determinants of the annual number of Pex (P < 0.01, with lower 25-OHD level and female gender associated with Pex). There was a significant influence of gender (P < 0.05) and a near-significant influence of serum 25-OHD (P < 0.08) on hospitalization rate. There was no effect of 25-OHD levels on PFTs. Other candidate factors (age, season, gender, pancreatic sufficiency, or severity of genetic mutation) did not significantly effect 25-OHD level. Conclusions: The annual number of Pex in pediatric CF patients is significantly associated with 25-OHD levels and gender, raising the consideration that maintaining vitamin D sufficiency may lead to decreased incidence of Pex and hospitalizations requiring antibiotic therapy. Pediatr Pulmonol. 2015;50:441–446. ß 2015 Wiley Periodicals, Inc. Key words: cystic fibrosis; vitamin D; pediatrics.
INTRODUCTION
Cystic fibrosis is a multi-system genetic disease characterized by defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which results in recurrent infective pulmonary exacerbations.1 Recent retrospective studies in adult CF patients have demonstrated positive correlations between serum 25hydroxyvitamin D (25-OHD) concentrations and lung function.2 Suggested mechanisms by which vitamin D improves lung function relate to its regulation of inflammatory mediators, and on recovery from respiratory infections. These processes are speculated to occur via enhanced innate immunity through up-regulation of antimicrobial peptides.3 The vitamin D receptor (VDR) is found in many tissues including small intestine, colon, osteoblasts, activated T and B-lymphocytes, islet cells, brain, heart, skin, gonads, prostate, breast, and mononuclear cells.4 The discovery that VDR is widely expressed, as well as accumulated data supporting a relationship between serum 25-OHD and chronic metabolic, cardiovascular, and neoplastic diseases have led to utilization of vitamin D supplementation for ß 2015 Wiley Periodicals, Inc.
potential prevention and treatment of numerous disorders, such as obesity, metabolic syndrome and type 2 diabetes mellitus.5 Chronic vitamin D deficiency in CF patients is a common problem, thought to be caused by impaired absorption of fat-soluble vitamins, decreased sunlight exposure, and suboptimal intake of vitamin D containing
1 Department of Pediatrics, Sections of Endocrinology, Yale University School of Medicine, New Haven, Connecticut. 2
Respiratory Medicine, New Haven, Connecticut.
3 Veterans Administration Cooperative Studies Program Coordinating Center, VA Connecticut Healthcare System, West Haven, Connecticut.
Correspondence to: Thomas Carpenter, MD, Yale University, PO Box 208064, New Haven, CT 06520-9064. E-mail: [email protected] Received 30 January 2014; Revised 16 November 2014; Accepted 30 November 2014. DOI 10.1002/ppul.23161 Published online 5 February 2015 in Wiley Online Library (wileyonlinelibrary.com).
442
Vanstone et al.
foods or supplements.6 Vitamin D supplementation may well have significant implications for patients with cystic fibrosis (CF), where in a recent study by Grossmann et al., a single bolus of cholecalciferol increased serum 25-OHD concentrations and was associated with a trend toward improved clinical outcomes in CF subjects hospitalized for a pulmonary exacerbation.7 In the setting of a pulmonary exacerbation, vitamin D is thought to improve the innate immune response to chronic pulmonary infections and inflammation, the most common causes of morbidity and mortality in CF. According to guidelines recently published by the Cystic Fibrosis Foundation (CFF) for the screening, management and diagnosis of vitamin D deficiency, sufficiency was defined as a serum 25-OHD level of 75 mol/l (30 ng/ml) based on previously published guidelines by the National Osteoporosis Foundation targeting markers of bone health and fracture risk.8 The CFF guidelines also stated that vitamin D status should be checked annually in all patients with CF, ideally at the end of winter.8 The purpose of this study was to examine the effect of vitamin D status in pediatric patients with cystic fibrosis on their annual frequency of pulmonary exacerbations, and to examine factors that may influence the ability of this patient population to maintain vitamin D sufficiency. Our hypothesis is that higher levels of serum 25-OHD would be associated with fewer Pex and hospitalizations, and improved lung function based on pulmonary function tests (PFTs). Clinical implications of an association between these clinical factors and vitamin D status may include use of vitamin D as treatment for acute pulmonary exacerbations, and guidance of target serum 25-OHD levels for minimizing chronic inflammation to prevent future exacerbations. METHODS Sample
We conducted a retrospective chart review of patients ages 5 through 22 years followed at the Cystic Fibrosis Care Center and Clinic at Yale-New Haven Hospital, New Haven, CT. Patients had given consent to participate in the Cystic Fibrosis Patient Registry, which reports patient data from CF patients who receive care at CF Foundationaccredited centers. The registry and its potential for unidentified data to be analyzed were approved by the appropriate IRB, the Yale Human Investigation Committee. Appropriate consent for the registry and data analysis as performed was obtained for all subjects whose data was accessed. Patients were included if they met minimum age requirement of 5 years (chosen because this age group can reliably perform pulmonary function testing), had at least four clinic visits and at least one 25-OHD measurement between January 1, 2011 and December 31, Pediatric Pulmonology
2011. Both pancreatic sufficient and insufficient patients were included in the study. Finally, we did not exclude patients taking vitamin D supplementation in doses greater than those recommended by the Cystic Fibrosis Foundation,4 as the goal of our study was to evaluate the impact of the 25-OHD level, not the dosing strategy. Data Collection
Patients’ records were used to obtain patient characteristics (age, gender, BMI, pancreatic sufficiency, genotype), pulmonary function test results, presence or absence of pancreatic sufficiency, vitamin D status, identified pulmonary exacerbations, and episodes of hospitalization. The pulmonary function test results were averaged for each visit during the year of the chart review. This time frame included both clinical stability as well as periods of illness. Genotypes were classified using 2 methods; the Class 1–3 class 4–5 strategy, and queried on line on the cftr2 database to confirm pancreatic status of each mutation.9,10 Of the 90 subjects, 80 have 2 alleles that fall into the Class 1–3 mutations (consistent with pancreatic insufficiency), 9 subjects have one class 1–3 mutation and one Class 4–5 mutation, suggesting they may be pancreatic sufficient or pancreatic variable, 1 subject has not been fully genotyped but has a positive 72 hr fecal fat collection and this improved on pancreatic enzymes and hence is assumed to be pancreatic insufficient. This categorization scheme also coincided with the clinical course of the subjects. Date of 25-OHD testing was assigned to a season for analysis as follows: Winter (January–March), Spring (April–June), Summer (July–September), and Fall (October–December). Serum 25-OHD levels were measured at the Yale Mineral Metabolism Laboratory using a radioimmunoassay kit (Diasorin, Stillwater, MN, USA). Results of samples are consistently in agreement with the midrange of outcomes reported with the use of this assay by the International Vitamin D External Quality Assessment Scheme (DEQAS) system11; the assay detects both 25-OHD2 and 25-OHD3. The inter- and intra-assay coefficients of variation for the 25-OHD assay are 9.6% and 6.6%, respectively. Pulmonary function tests (PFTs) included were forced expiratory volume in one second (FEV1), flow between 25 and 75 percent of the vital capacity (FEF25–75), FEV1 and FEF25–75 percent adjusted for age, and forced vital capacity (FVC). Identification of pulmonary exacerbations employed 2 complementary methods: first, the initiation or increase of antibiotic dosages, based on reported symptoms, was defined as an exacerbation. A second method employed the Akron Pulmonary Exacerbation Score12; a comprehensive assessment incorporating previously published exacerbation criteria and associated symptoms.13 This score is divided into three areas:
Vitamin D Determines Pulmonary Exacerbations
systemic signs and symptoms, pulmonary signs and symptoms, and objective measurements (Table 1), as obtained during a routine CF clinic visit. Thus a pulmonary exacerbation was identified by either new or increased use of antibiotics, or an Akron Pulmonary Exacerbation threshold score of 5. Both methods were employed as to capture exacerbations that did not occur during regularly scheduled clinic visits, and thus reflecting overall health status more accurately. Statistical Analysis
Descriptive statistics were used to summarize the data. Multivariate regression analysis was performed as to determine specific significant determinants of number of pulmonary exacerbations and pulmonary function test. In the regression model the total number of reported pulmonary exacerbations serves as a dependent variable and 25-hydroxyvitamin D, gender, pancreatic sufficient status and genetic mutation status serve as independent variables. The same analysis was performed on pulmonary function test. There is no elimination or selection in the modeling process. All independent variables are included and adjusted in the reported results. We used a 0.05 significance level for all test. SAS (version 9.2) was employed for all analyses. RESULTS Profile of Subjects
The medical record of all patients encountered at the pediatric Cystic Fibrosis Care Center and Clinic at Yale-
443
New Haven Hospital from January 1st 2011 to December 31st, 2011 were reviewed (total 89); 53 patients were included in the study. Of the 36 who were excluded 14 had transferred to a clinic for adults during the time frame of the study, 13 were less than 5 years of age, 5 had moved, and 4 did not meet inclusion criteria of 4 clinic visits in the one year period indicated above. Table 2 shows patient characteristics for the entire sample. Patients ranged in age from 5 to 22 years, and approximately onehalf of the subjects were male. Most patients manifest pancreatic insufficiency (80%). The median BMI was 50th centile for age; 4 patients had a BMI less than the 5th centile. The mean serum 25-OHD level was 69 nmol/l (27.5 ng/ml), and the range was 22–135 nmol/l. Seventy percent of subjects had a serum 25-OHD level less than 75 nmol/l (30 ng/ml) and 12% of subjects had serum 25OHD levels less than 50 nmol/l (20 ng/ml) (Fig. 1). Pulmonary Exacerbations and Function Tests
There were a total of 155 pulmonary exacerbations occurring in the 53 patients in the identified year of analysis (Table 3). Of these, 33 were admitted to the hospital and treated with intravenous antibiotics, 119 were treated with oral antibiotics as outpatients (65 were seen in clinic, 54 were treated based on symptoms described via telephone encounter with the patient and/or caregiver). Examination of vitamin D levels showed that 38 subjects had levels less than 75 nmol/l (73% of the total number of subjects), and would be considered deficient by current guidelines. This group had 130 (83%) of the exacerbations, and those with levels >75 nmol/l (14
TABLE 1— The Akron Pulmonary Exacerbation Score Systemic Symptoms/Signs Fever > 38C in the prior 2 weeks? Malaise or fatigue in the prior 2 weeks? Increased or new school absences in the prior 2 weeks? Anorexia or poor appetite in the prior 2 weeks? Weight loss (5%) of poor weight gain compared to last clinic visit (or in the last 3 months) ? Pulmonary Symptoms/ Signs Increased cough (frequency, intensity, duration) for 1 week? Major change in sputum or change in chest congestion for 1 week? Increased DOE or SOB at rest? Change in chest exam or increased WOB or respiratory rate? Objective Measurements Decrease in FEV1 (compared to highest value of the prior 6 months) ? New chest radiograph abnormality?
Hemoptysis? Decreased SaO2 from baseline (compared to highest value of the prior 6 months) ?
Yes ¼ 1 Yes ¼ 1 Yes ¼ 2 Yes ¼ 1 Yes ¼ 2
No ¼ 0 No ¼ 0 No ¼ 0 No ¼ 0 No ¼ 0
None ¼ 1 Mild ¼ 1 Significant ¼ 2 None ¼ 1 Mild ¼ 1 Significant ¼ 2 Yes ¼ 2 No ¼ 0 Yes ¼ 2 No ¼ 0
Association Between Serum 25-Hydroxyvitamin D Level and Pulmonary Exacerbations in Cystic Fibrosis Michelle B. Vanstone, MD,1 Marie E. Egan, MD,2 Jane H. Zhang, and Thomas O. Carpenter, MD1*
PhD,
3
Summary. Objective: To identify effects of vitamin D status, as defined by circulating 25hydroxyvitamin D (25-OHD) levels on the annual frequency of pulmonary exacerbations (Pex) and hospitalizations, on standard measures of pulmonary function, and to identify determinants of 25-OHD levels in pediatric patients with cystic fibrosis (CF). Hypothesis: Higher levels of serum 25-OHD would be associated with fewer Pex and hospitalizations, and improved lung function based on pulmonary function tests (PFTs). Study design: Retrospective chart review of 53 pediatric patients from January 2011 to December 2011. Significant relationships were examined using linear and logistic regression analyses. Patient Selection: Patients ages 5 through 22 years followed at the CF Care Center and Clinic at Yale-New Haven Hospital, New Haven, CT., who had at least four clinic visits and at least one 25-OHD measurement between January 1, 2011 and December 31, 2011. Results: Serum 25-OHD level and gender were strong independent determinants of the annual number of Pex (P < 0.01, with lower 25-OHD level and female gender associated with Pex). There was a significant influence of gender (P < 0.05) and a near-significant influence of serum 25-OHD (P < 0.08) on hospitalization rate. There was no effect of 25-OHD levels on PFTs. Other candidate factors (age, season, gender, pancreatic sufficiency, or severity of genetic mutation) did not significantly effect 25-OHD level. Conclusions: The annual number of Pex in pediatric CF patients is significantly associated with 25-OHD levels and gender, raising the consideration that maintaining vitamin D sufficiency may lead to decreased incidence of Pex and hospitalizations requiring antibiotic therapy. Pediatr Pulmonol. 2015;50:441–446. ß 2015 Wiley Periodicals, Inc. Key words: cystic fibrosis; vitamin D; pediatrics.
INTRODUCTION
Cystic fibrosis is a multi-system genetic disease characterized by defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which results in recurrent infective pulmonary exacerbations.1 Recent retrospective studies in adult CF patients have demonstrated positive correlations between serum 25hydroxyvitamin D (25-OHD) concentrations and lung function.2 Suggested mechanisms by which vitamin D improves lung function relate to its regulation of inflammatory mediators, and on recovery from respiratory infections. These processes are speculated to occur via enhanced innate immunity through up-regulation of antimicrobial peptides.3 The vitamin D receptor (VDR) is found in many tissues including small intestine, colon, osteoblasts, activated T and B-lymphocytes, islet cells, brain, heart, skin, gonads, prostate, breast, and mononuclear cells.4 The discovery that VDR is widely expressed, as well as accumulated data supporting a relationship between serum 25-OHD and chronic metabolic, cardiovascular, and neoplastic diseases have led to utilization of vitamin D supplementation for ß 2015 Wiley Periodicals, Inc.
potential prevention and treatment of numerous disorders, such as obesity, metabolic syndrome and type 2 diabetes mellitus.5 Chronic vitamin D deficiency in CF patients is a common problem, thought to be caused by impaired absorption of fat-soluble vitamins, decreased sunlight exposure, and suboptimal intake of vitamin D containing
1 Department of Pediatrics, Sections of Endocrinology, Yale University School of Medicine, New Haven, Connecticut. 2
Respiratory Medicine, New Haven, Connecticut.
3 Veterans Administration Cooperative Studies Program Coordinating Center, VA Connecticut Healthcare System, West Haven, Connecticut.
Correspondence to: Thomas Carpenter, MD, Yale University, PO Box 208064, New Haven, CT 06520-9064. E-mail: [email protected] Received 30 January 2014; Revised 16 November 2014; Accepted 30 November 2014. DOI 10.1002/ppul.23161 Published online 5 February 2015 in Wiley Online Library (wileyonlinelibrary.com).
442
Vanstone et al.
foods or supplements.6 Vitamin D supplementation may well have significant implications for patients with cystic fibrosis (CF), where in a recent study by Grossmann et al., a single bolus of cholecalciferol increased serum 25-OHD concentrations and was associated with a trend toward improved clinical outcomes in CF subjects hospitalized for a pulmonary exacerbation.7 In the setting of a pulmonary exacerbation, vitamin D is thought to improve the innate immune response to chronic pulmonary infections and inflammation, the most common causes of morbidity and mortality in CF. According to guidelines recently published by the Cystic Fibrosis Foundation (CFF) for the screening, management and diagnosis of vitamin D deficiency, sufficiency was defined as a serum 25-OHD level of 75 mol/l (30 ng/ml) based on previously published guidelines by the National Osteoporosis Foundation targeting markers of bone health and fracture risk.8 The CFF guidelines also stated that vitamin D status should be checked annually in all patients with CF, ideally at the end of winter.8 The purpose of this study was to examine the effect of vitamin D status in pediatric patients with cystic fibrosis on their annual frequency of pulmonary exacerbations, and to examine factors that may influence the ability of this patient population to maintain vitamin D sufficiency. Our hypothesis is that higher levels of serum 25-OHD would be associated with fewer Pex and hospitalizations, and improved lung function based on pulmonary function tests (PFTs). Clinical implications of an association between these clinical factors and vitamin D status may include use of vitamin D as treatment for acute pulmonary exacerbations, and guidance of target serum 25-OHD levels for minimizing chronic inflammation to prevent future exacerbations. METHODS Sample
We conducted a retrospective chart review of patients ages 5 through 22 years followed at the Cystic Fibrosis Care Center and Clinic at Yale-New Haven Hospital, New Haven, CT. Patients had given consent to participate in the Cystic Fibrosis Patient Registry, which reports patient data from CF patients who receive care at CF Foundationaccredited centers. The registry and its potential for unidentified data to be analyzed were approved by the appropriate IRB, the Yale Human Investigation Committee. Appropriate consent for the registry and data analysis as performed was obtained for all subjects whose data was accessed. Patients were included if they met minimum age requirement of 5 years (chosen because this age group can reliably perform pulmonary function testing), had at least four clinic visits and at least one 25-OHD measurement between January 1, 2011 and December 31, Pediatric Pulmonology
2011. Both pancreatic sufficient and insufficient patients were included in the study. Finally, we did not exclude patients taking vitamin D supplementation in doses greater than those recommended by the Cystic Fibrosis Foundation,4 as the goal of our study was to evaluate the impact of the 25-OHD level, not the dosing strategy. Data Collection
Patients’ records were used to obtain patient characteristics (age, gender, BMI, pancreatic sufficiency, genotype), pulmonary function test results, presence or absence of pancreatic sufficiency, vitamin D status, identified pulmonary exacerbations, and episodes of hospitalization. The pulmonary function test results were averaged for each visit during the year of the chart review. This time frame included both clinical stability as well as periods of illness. Genotypes were classified using 2 methods; the Class 1–3 class 4–5 strategy, and queried on line on the cftr2 database to confirm pancreatic status of each mutation.9,10 Of the 90 subjects, 80 have 2 alleles that fall into the Class 1–3 mutations (consistent with pancreatic insufficiency), 9 subjects have one class 1–3 mutation and one Class 4–5 mutation, suggesting they may be pancreatic sufficient or pancreatic variable, 1 subject has not been fully genotyped but has a positive 72 hr fecal fat collection and this improved on pancreatic enzymes and hence is assumed to be pancreatic insufficient. This categorization scheme also coincided with the clinical course of the subjects. Date of 25-OHD testing was assigned to a season for analysis as follows: Winter (January–March), Spring (April–June), Summer (July–September), and Fall (October–December). Serum 25-OHD levels were measured at the Yale Mineral Metabolism Laboratory using a radioimmunoassay kit (Diasorin, Stillwater, MN, USA). Results of samples are consistently in agreement with the midrange of outcomes reported with the use of this assay by the International Vitamin D External Quality Assessment Scheme (DEQAS) system11; the assay detects both 25-OHD2 and 25-OHD3. The inter- and intra-assay coefficients of variation for the 25-OHD assay are 9.6% and 6.6%, respectively. Pulmonary function tests (PFTs) included were forced expiratory volume in one second (FEV1), flow between 25 and 75 percent of the vital capacity (FEF25–75), FEV1 and FEF25–75 percent adjusted for age, and forced vital capacity (FVC). Identification of pulmonary exacerbations employed 2 complementary methods: first, the initiation or increase of antibiotic dosages, based on reported symptoms, was defined as an exacerbation. A second method employed the Akron Pulmonary Exacerbation Score12; a comprehensive assessment incorporating previously published exacerbation criteria and associated symptoms.13 This score is divided into three areas:
Vitamin D Determines Pulmonary Exacerbations
systemic signs and symptoms, pulmonary signs and symptoms, and objective measurements (Table 1), as obtained during a routine CF clinic visit. Thus a pulmonary exacerbation was identified by either new or increased use of antibiotics, or an Akron Pulmonary Exacerbation threshold score of 5. Both methods were employed as to capture exacerbations that did not occur during regularly scheduled clinic visits, and thus reflecting overall health status more accurately. Statistical Analysis
Descriptive statistics were used to summarize the data. Multivariate regression analysis was performed as to determine specific significant determinants of number of pulmonary exacerbations and pulmonary function test. In the regression model the total number of reported pulmonary exacerbations serves as a dependent variable and 25-hydroxyvitamin D, gender, pancreatic sufficient status and genetic mutation status serve as independent variables. The same analysis was performed on pulmonary function test. There is no elimination or selection in the modeling process. All independent variables are included and adjusted in the reported results. We used a 0.05 significance level for all test. SAS (version 9.2) was employed for all analyses. RESULTS Profile of Subjects
The medical record of all patients encountered at the pediatric Cystic Fibrosis Care Center and Clinic at Yale-
443
New Haven Hospital from January 1st 2011 to December 31st, 2011 were reviewed (total 89); 53 patients were included in the study. Of the 36 who were excluded 14 had transferred to a clinic for adults during the time frame of the study, 13 were less than 5 years of age, 5 had moved, and 4 did not meet inclusion criteria of 4 clinic visits in the one year period indicated above. Table 2 shows patient characteristics for the entire sample. Patients ranged in age from 5 to 22 years, and approximately onehalf of the subjects were male. Most patients manifest pancreatic insufficiency (80%). The median BMI was 50th centile for age; 4 patients had a BMI less than the 5th centile. The mean serum 25-OHD level was 69 nmol/l (27.5 ng/ml), and the range was 22–135 nmol/l. Seventy percent of subjects had a serum 25-OHD level less than 75 nmol/l (30 ng/ml) and 12% of subjects had serum 25OHD levels less than 50 nmol/l (20 ng/ml) (Fig. 1). Pulmonary Exacerbations and Function Tests
There were a total of 155 pulmonary exacerbations occurring in the 53 patients in the identified year of analysis (Table 3). Of these, 33 were admitted to the hospital and treated with intravenous antibiotics, 119 were treated with oral antibiotics as outpatients (65 were seen in clinic, 54 were treated based on symptoms described via telephone encounter with the patient and/or caregiver). Examination of vitamin D levels showed that 38 subjects had levels less than 75 nmol/l (73% of the total number of subjects), and would be considered deficient by current guidelines. This group had 130 (83%) of the exacerbations, and those with levels >75 nmol/l (14
TABLE 1— The Akron Pulmonary Exacerbation Score Systemic Symptoms/Signs Fever > 38C in the prior 2 weeks? Malaise or fatigue in the prior 2 weeks? Increased or new school absences in the prior 2 weeks? Anorexia or poor appetite in the prior 2 weeks? Weight loss (5%) of poor weight gain compared to last clinic visit (or in the last 3 months) ? Pulmonary Symptoms/ Signs Increased cough (frequency, intensity, duration) for 1 week? Major change in sputum or change in chest congestion for 1 week? Increased DOE or SOB at rest? Change in chest exam or increased WOB or respiratory rate? Objective Measurements Decrease in FEV1 (compared to highest value of the prior 6 months) ? New chest radiograph abnormality?
Hemoptysis? Decreased SaO2 from baseline (compared to highest value of the prior 6 months) ?
Yes ¼ 1 Yes ¼ 1 Yes ¼ 2 Yes ¼ 1 Yes ¼ 2
No ¼ 0 No ¼ 0 No ¼ 0 No ¼ 0 No ¼ 0
None ¼ 1 Mild ¼ 1 Significant ¼ 2 None ¼ 1 Mild ¼ 1 Significant ¼ 2 Yes ¼ 2 No ¼ 0 Yes ¼ 2 No ¼ 0
