Objective: The objective of the present study is to compare daily weight gain and laboratory analysis (72-hour fecal fat and steatocrit) with fecal elastase-1 (EL-1) when diagnosing pancreatic insufficiency (PI) in infants with cystic fibrosis (CF).
Methods: A total of 39 infants with CF, diagnosed consecutively by newborn screening at 2 referral centers, were included in the study. Daily weight gain and results of laboratory analysis of stool samples were compared using the κ coefficient and the receiver operator characteristic (ROC) curve.
Results: Using the criterion of low daily weight gain, the frequency of PI was 92.3%; using the 72-hour fecal fat, steatocrit, and fecal EL-1 tests, the frequency was 42.3%, 86.2%, and 84.6%, respectively. EL-1 was used as the reference test. It was observed that the criteria of low daily weight gain (<50th percentile) and abnormal steatocrit, used together, showed the highest sensitivity (91.3%) and specificity (83.3%) for the diagnosis of PI.
Conclusions: When fecal EL-1 analysis is not immediately available, low daily weight gain associated with abnormal steatocrit can be adopted as a criterion for initiating pancreatic enzyme replacement therapy in infants with CF; however, EL-1 testing should be performed later for confirmation of PI.
*University Hospital/Federal University of Minas Gerais, Belo Horizonte
†Health Sciences Center, Federal University of Saõ João del-Rei, Divinópolis
‡Department of Pediatrics, Diagnostic Support Action and Research Center (NUPAD)/Medical School, Pediatric Gastroenterology Center/University Hospital–Federal University of Minas Gerais
§University Hospital, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Address correspondence and reprint requests to Elizabet V Guimarães, MD, PhD, Associate Professor, Departamento de Pediatria, Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil (e-mail: email@example.com).
Received 26 October, 2011
Accepted 9 August, 2012
P.C. is supported by the Brazilian research agencies CNPq-Brazilian Council for Scientific and Technological Development (Grant #303827/2009-2) and FAPEMIG—Minas Gerais State Foundation for Research Support (Grant #PPM-00230-10).
The authors report no conflict of interest.
See “Assessing Exocrine Pancreatic Function: When the Best Test Is Not Possible” by Baker on page 116.
In patients with cystic fibrosis (CF), signs of pancreatic insufficiency (PI) occur early. PI is seen in approximately 90% of patients with CF by the end of the first year of life, with 60% showing symptoms at birth (1). Infants with PI present with chronic diarrhea, malabsorption, steatorrhea, and an insatiable appetite, as well as poor weight gain with varying degrees of malnutrition; however, studies are scarce on the accuracy of clinical PI diagnosis during the first weeks of life, and studies regarding PI laboratory methods for this age group are inconclusive.
Both direct and indirect laboratory methods may be used for the diagnosis of exocrine PI. The direct methods are invasive and are uncommon in clinical practice. Even though the secretin-cholecystokinin test is invasive, and thus inadequate for routine pediatric evaluation, it remains the criterion standard for the detection of PI. However, 72-hour fecal fat, the steatocrit, and fecal elastase-1 (EL-1) are methods that can indirectly assess PI. Among these, the 72-hour fecal fat is well recognized as the criterion standard for the determination of fat absorption (2); its main problem is related to the mandatory 72-hour stool collection. Conversely, the steatocrit is a simple semiquantitative method of assessing fat absorption. Phuapradit et al (3) proposed this method in 1981 for measuring the fecal fat in newborns. EL-1 is an enzyme excreted by the pancreas that is neither degraded in the gastrointestinal tract nor affected by pancreatic enzyme replacement therapy (PERT) (4). EL-1 estimation requires a small single stool sample. It shows high sensitivity and specificity (5,6) when compared with the secretin-cholecystokinin test; however, it costs >72-hour fecal fat or steatocrit testing. EL-1 is unreliable during the first 2 weeks after birth and in the presence of diarrhea (7,8).
The early and accurate identification of PI will improve the effectiveness of PERT and the nutritional status of affected infants. In the present study, we compared EL-1 to 72-hour fecal fat, steatocrit, and daily weight gain with the aim of identifying a criterion for the diagnosis of PI in infants with CF when EL-1 is not immediately available.
The participants were patients diagnosed by the Newborn Screening Program of Minas Gerais State from December 2006 to December 2008. The screening adopts a 3-step strategy (immunoreactive trypsin (IRT)/IRT/sweat test). The sweat test was performed according to the Gibson-Cooke method. During this period, 45 infants with CF were diagnosed; 39 were included in the study. Of the patients not included, 3 were severely malnourished and were administered pancreatic enzyme immediately after the diagnosis. The discontinuation of PERT, which was mandatory for performing steatocrit and 72-hour fecal fat testing, was contraindicated for them owing to the severity of their nutritional status. Two patients with ileostomy because of meconium ileus were not included. One patient's guardian did not provide consent. For patients who had nutritionally recovered, pancreatic enzyme was discontinued 3 days before the stool sample collection and during the collection.
The following variables were recorded in the study form: sex, F508del status, birth weight, age and weight at CF diagnosis, and age and type of feeding when the laboratory tests were performed (breast-feeding, formula feeding, breast and formula feeding, or complementary food).
Specific instructions about the stool sample collection and storage were given to the infants’ parents or legal guardians, both verbally and in writing. The stool samples included a full 72-hour stool sample (to measure the 72-hour fecal fat), as well as 2 extremely small stool samples from a random defecation from the 72-hour collection (to measure the steatocrit and EL-1). The EL-1 stool samples were stored in a domestic refrigerator (temperature ranging from 2°C to 8°C, checked daily) and transported in coolers on ice to the laboratory, where they were stored in a freezer at −17°C until they were assayed.
Clinical Diagnosis of PI
The clinical criterion adopted for the clinical diagnosis of PI was mean daily weight gain. The 50th percentile of daily weight gain as found by Guo et al (9) was adopted as a reference for “insufficient daily weight gain.”
Laboratory Diagnosis of PI
The 72-hour fecal fat analysis was performed according to the van de Kamer method (10). This method has been routinely used at our center for the last 30 years and was standardized by Penna et al (11). These authors developed reference values based on a patient's diet type to classify test results as either normal or abnormal. The upper limits are as follows: 0.93 g of fat/day for children younger than 6 months who are exclusively breast-fed, 2.25 g of fat/day for children younger than 6 months who are fed formula or cow's milk, and 2.28 g of fat/day for children older than 6 months, regardless of the diet.
The steatocrit was measured according to the technique described by Phuapradit et al (3). Reference values for the classification of normal and abnormal values were based on patient age. (12). Values included 4.04% (age 0–1 month), 1.38% (age 1–3 months), and 0.29% (age 3–72 months).
The EL-1 was performed using the sandwich-enzyme-linked immunosorbent assay method, with 2 specific monoclonal antibodies for human pancreatic EL-1. The assays were performed in duplicate in accordance with the technique recommended by the manufacturer (ScheBo Biotech Ag, Giessen, Germany) and by others (13,14). PI was defined when EL-1 was <200 μg/g (1).
EL-1 was chosen as the reference test and was compared with 72-hour fecal fat, steatocrit, and 50th percentile of daily weight gain using the κ coefficient and the receiver operating characteristic (ROC) curve. Therefore, the sensitivity, specificity, positive predictive value, and negative predictive value for each test and for the clinical criterion were obtained. A P value of <0.05 was adopted as the significance level. Statistical analysis was computed using R software version 2.7.1 (R software, Free Software Foundation, Boston, MA) and EpiInfo version 6.04 (Centers for Disease Control and Prevention, Atlanta, GA).
The study protocol and informed consent were approved by the research ethics committees at Universidade Federal de Minas Gerais and Fundação Hospitalar do Estado de Minas Gerais. The written consent form was signed in duplicate by the patient's parent or a legally responsible guardian.
Table 1 displays the general characteristics of the studied subjects. The mean age at diagnosis was 42 days (SD ± 23 days). The mean weight gain from birth until the day of CF diagnosis was recorded for all of the subjects and was equal to 13.87 g/day (SD ± 11.50 g/day). Most of the patients (56.4%) were exclusively breast-fed (Table 1). All 39 patients underwent EL-1 analysis. The steatocrit was performed in 29 (74%) patients and the 72-hour fecal fat in 26 (67%) of them.
Table 2 shows the PI diagnosis distribution for each of the 3 laboratory tests and for the weight gain clinical criterion. Figure 1 illustrates the ROC curve of EL-1 versus the lab tests, the clinical criterion, and the criterion of “daily weight gain below the 50th percentile associated with abnormal steatocrit.”
When we added the steatocrit to the weight gain (as reported above), the area under the curve increased from 0.627 to 0.873, meaning that for our patients this strategy was more accurate than every other criterion alone.
Table 3 presents the sensitivity, specificity, positive predictive value, and negative predictive value data for the laboratory tests, the clinical criterion (daily weight gain below the 50th percentile), and the strategy “daily weight gain below the 50th percentile associated with abnormal steatocrit” when compared with those of EL-1.
The diagnosis of PI in newborns and extremely young children is rarely a simple task. Numerous efforts have been made to determine which indirect laboratory method is most adequate for diagnosing PI in patients with CF (2,15–19). Clinical criteria such as diarrheal stool and insatiable appetite are nonspecific and thus inadequate to characterize PI. Direct pancreatic function tests, although the criterion standard, are cumbersome and not widely available. No study was found that evaluated daily weight gain as a sole criterion for PI in infants with CF.
Recent studies have assessed the role of EL-1 in the diagnosis of PI, especially in patients with CF. In patients with PI, Löser et al (5) compared secretin-cholecystokinin (the criterion standard test for PI) with EL-1 and found a sensitivity and specificity of 93%, with a cutoff point of 200 μg/g. Another study found 94% sensitivity for patients with severe PI. EL-1 testing detected only 82% of patients with steatorrhea and was less sensitive for mild PI (20); however, EL-1 has been widely accepted as the diagnostic test of choice because duodenal intubation is seldom performed and other tests have proven to be inferior. Therefore, EL-1 was adopted as the reference test for the present study, and PI was defined as EL-1 <200 μg/g; however, all of the patients with PI presented with EL-1 <100 μg/g.
EL-1 and 72-hour fecal fat show agreement with low sensitivity (50%) and extremely high specificity (100%) in the diagnosis of severe PI (EL-1 <100 mg/g). Walkowiak (21) compared EL-1 (EL-1 <200 mg/g) with 72-hour fecal fat testing in patients with CF with either pancreatic sufficiency or PI. The EL-1 exhibited sensitivity of 98.7% for patients with PI; however, the sensitivity was only 50% in the CF group with pancreatic sufficiency.
In the present study, we performed the steatocrit by collecting an extremely low volume of single stool sample. Previously, Rawashdeh et al and Guarino et al (22,23) had demonstrated only a small degree of variability between the multiple stool sample method and the single stool sample method; therefore, the single stool sample method was chosen for its simplicity.
Conflicting results have also been obtained in studies that evaluated the correlation between EL-1 and steatocrit in PI. In the evaluation of pancreatic function in patients with HIV, Carroccio et al (24) found a statistically significant correlation between the tests (P < 0.03). Likewise, Mancilla et al (25) found a significant association between a low EL-1 level and steatocrit in diabetic adults. In another study, however, Carroccio et al (16) did not find any correlation between EL-1 and steatocrit in patients with CF. In our study, a moderate agreement between the tests was found. The variability across those findings may be explained by a multiplicity of factors that cause steatorrhea in patients with CF and by differences in the diets and ages of the studied individuals. Although steatocrit testing may be effective for young infants, this effectiveness may not be applicable to older children with CF, in whom the dispersion of the fat in the stool is much less homogeneous. Therefore, continued caution is recommended while using this test in the older age group.
An agreement between EL-1 level and the 50th percentile of daily weight gain was found in the present study, with high correlation (96.9%) but low specificity (28.6%). Based on the hypothesis that the clinical parameter (50th percentile weight gain) could be an adequate screening threshold for PI, analyses were conducted associating this parameter with laboratory tests. The criterion “weight gain <50th percentile associated with abnormal steatocrit” showed a moderate correlation with adequate sensitivity and specificity compared with EL-1 (Table 3). The routine evaluation of newborns with CF at the time of diagnosis includes the recording of their weight followed by the evaluation of their daily weight gain. Infants with CF that are <50th percentile on the daily weight gain curve should be considered as most likely having PI. The immediate performance of the steatocrit assay on these newborns should be considered in every center that assists these children, if EL-1 is not immediately available. The steatocrit is a low-cost method that involves a single stool sample collection and tasks that are simple to execute; however, infants presenting with extremely low percentiles of daily weight gain (<10th percentile) must be assessed with additional caution because the specificity of this criterion increases as the percentile decreases. These infants are most likely to benefit immediately from prescribed enzymes; however, when weight gain is a less urgent concern, it may be wise to remember that prescribed enzymes may pose unnecessary risks and increase the caregiver's workload. Moreover, it is worth debating whether an excessive workload for the caregiver soon after diagnosis could negatively affect the caregiver's bond with the child.
Some factors may limit the general results of the present study. First, in the initial phase of the study, 33% of the samples collected for the 72-hour fecal fat were excluded because they were considered inadequate at the time of collection (extremely low total stool weight), demonstrating the difficulty of collecting stool from infants with CF. Second, regarding the steatocrit, 10 samples were lost during the pilot study. Finally, EL-1 is not the criterion standard test for PI, and the results obtained were not compared with those of the secretin-cholecystokinin test. These comparisons would most likely demonstrate more accurately the value of the tests and the strategy analyzed here.
We concluded that the 50th percentile of daily weight gain, when associated with abnormal steatocrit, could be adopted as the criterion for the immediate prescription of pancreatic enzymes in centers where tests for EL-1 are not immediately available. If steatocrit testing is not feasible, daily weight gain should be closely monitored. If the patient is below the 10th percentile of daily weight gain or demonstrates a progressive decline in percentile, a trial with PERT should be initiated immediately; however, EL-1 should be performed later for confirmation of PI.
The authors are grateful to the Hospital João Paulo II's team for providing patients for the present study and to the CEAPS team for their support. The authors also thank the Pró-reitoria de Pesquisa da Universidade Federal de Minas Gerais for supporting English-language review.
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Keywords:Copyright 2013 by ESPGHAN and NASPGHAN
body weight; cystic fibrosis; diagnosis; exocrine pancreatic insufficiency; infants