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Use Of
A Binomial Model To Predict A Lower Confidence Limit For Copper Deficiency
Prevalence In Feeder Calves
Ronald K. Tessman, DVM^{*} Jeff W. Tyler, DVM, PhD^{*} Stan W. Casteel, DVM, PhD^{} Robert L. Larson, DVM, PhD^{*} Jeff Lakritz, DVM, PhD^{*} Gary F. Krause, PhD James E. Williams, PhD§ Departments of ^{*}Veterinary
Medicine and Surgery, ^{}Veterinary Pathobiology, Statistics, § The described research was supported in part by grants
from The Committee On Research, College of Veterinary Medicine, University
of MissouriColumbia; the University of Missouri Agricultural Experiment
Station; and USDA Formula Funds. Additional support was provided by
the Minority Biomedical Researchers Training Initiative and the University
of Missouri Chancellors Gus T. Ridgel Fellowship for Underrepresented
Minority Americans. KEY WORDS: prevalence, binomial, Abstract The purpose of this study was to investigate a novel approach
to estimate a lower confidence limit for copper deficiency prevalence
using serum copper concentrations, which were based on the binomial
distribution. Paired liver and serum samples were collected from 33
calves. The binomial distribution was used to calculate the probability
of k positive test results in n trials at varying prevalences. This
process provided a de facto hypothesis test for the lower 95% confidence
limit of prevalence. For model validation, random samples of either
10 or 15 were drawn from the 33 serum samples. These samples were used
to compare the lower limit of prevalence using the developed model and
traditional methods. Results of the model verification trials are as
follows. Mean and standard deviation of the 95% lower confidence limit
of prevalence of the 10 and 15 sample trials were 0.49 ± 0.24 and 0.64 ± 0.19,
respectively. Using the Zdistribution for population proportions, the
mean and standard deviation of the 95% lower confidence limit of the
10 and 15sample trials were 0.95 ±
0.14 and 0.99 ± 0.06, respectively. The binomial model provided
a more satisfactory method to interpret imperfect test results than
the Zdistribution for population proportions. The described technique
has merit beyond the topic of copper deficiency. Rather than discount
imperfect tests, we envision using this procedure to develop confidence
limits for population prevalence in those instances in which test performance
(either sensitivity or specificity) of diagnostic tests is suboptimal. INTRODUCTION Ideally, diagnostic tests have high sensitivity and specificity. When tests are used to direct the medical management of individuals, the accuracy of individual test results is paramount. Additionally researchers, clinicians, and public health professionals are often asked to provide estimates of disease prevalence based on results of imperfect tests. Reasonable conclusions regarding the population disease behavior may be made using tests with lessthanoptimal sensitivity and specificity. Traditionally, real prevalence is calculated using the following formula: Equation (Eq) 1: where RP is real prevalence, AP is the apparent prevalence or proportion of positive test results, S_{p} is the specificity or likelihood of a negative test in a disease negative individual, and S_{e} is the sensitivity or likelihood of a positive test in a disease positive individual. It is possible to use the Zdistribution for population proportions to construct lower confidence intervals for calculated real prevalence using the following formula: 95% lower confidence limit of prevalence = RP 1.96 χ(RP(1RP)/n)2 where RP = calculated real prevalence, and n = sample size. It should be noted that small sample sizes produce broad confidence intervals that have little value in describing population prevalence.^{ } The purpose of this study was to develop an alternate approach based on the binomial probability distribution. For illustrative purposes, we examined detection of copper deficiency in cattle using serum copper concentrations. This procedure has previously been reported to have imperfect sensitivity (0.53) and specificity (0.89).3 True copper status was determined by liver copper concentrations. MATERIALS AND METHODS Theoretical Reasoning The hypothesis statement developed to guide model development was as follows: H_{0}: H_{a}:
It is intuitive that apparent prevalence equals the probability of a positive test result: AP = Pr{T+}.4 Eq 1 can be solved for apparent prevalence to obtain equation 2: Equation (Eq) 2: The binomial distribution requires an outcome which is either positive or negative and mutually exclusive; Pr{T+} = 1 Pr{T}, where Pr{T+} is the probability of a positive test and Pr{T} is the probability of a negative test. The reported sensitivity and specificity were used to calculate the Pr{T+} at all possible prevalences. These probabilities were used with various combinations of trials and successes in the binomial distribution equation to calculate the probability of k positive test results (serum copper £ 0.45 mg/mL) when n subjects are sampled at varying prevalences. The binomial distribution may be defined as follows: ^{ } Equation (Eq) 3: where P(X=k) is the probability of an event, n is the number of trials, k is the number of positive outcomes and p is the probability of a success in each trial. In our example, n was the number of calves sampled, k was the number of animals with a serum copper concentration less than or equal to 0.45 mg/g, and p was the hypothesized apparent prevalence as calculated using Eq 2. The event predicted was the likelihood of the number of positive tests being greater than or equal to k when n calves were sampled in a population of defined prevalence. If the calculated probability was very low, we assumed that the prevalence is higher than that which was previously hypothesized. The probability statement may be expressed as, Pr{RP ³ x} £ P value. Because this model was based on apparent prevalence, an additional probability statement was required. This statement may be expressed in the form of Pr{AP ³ y} £ P value. Therefore, the final probability statement required to reject hypotheses regarding prevalence based on randomly sampled data sets was as follows: Equation (Eq) 4: When the calculated Pr{AP ³ y}£ 0.025 we rejected the null hypothesis that real prevalence is less than an a priori hypothesized prevalence. We chose a p value £ 0.025 because it is equivalent to the lower limit of a 95% confidence interval. With the aid of a computer program (Microsoft Excel 2000, Seattle, WA), a large table representing the probability of all possible outcomes for trials consisting of greater than or equal to 5 and less than or equal to 20 samples at all possible real prevalences was produced. Model Validation Serum copper determinations were performed on samples taken from calves ranging in age from 6 to 9 months from a single herd. Paired liver and serum samples were collected from 33 calves. Blood was collected into evacuated tubes specifically manufactured for trace mineral determinations (Becton Dickinson and Company, Franklin Lakes, NJ). Liver biopsies were collected by transthoracic technique using a 16 gauge biopsy needle (Jorgensen Laboratories, Loveland, CO).6 All copper determinations were made through the use of atomic absorption spectrophotometry (PerkinElmer 2380, Norwal, CT) (wavelength, 324.7 nm) with a previously described method.3 The apparent prevalence
(AP) was calculated by dividing the number of positive tests, those
with a serum copper concentration less than or equal to 0.45 mg/g, by the total number of calves sampled at each
time period. Samples of sets of
10 and 15 serum copper results were randomly drawn without replacement
from the 33 serum copper determinations. For samples of each size (n
= 10 or 15), 1000 random sampling iterations were performed using a
computer software program (SPlus 2000, Mathsoft Inc., Seattle, WA).^{
}The number of positive test results (serum copper £ 0.45 mg/g) was determined for each sample set. Given the
number of positive tests (k) and the sample size (n = 10 or 15), the
95% lower confidence limit for prevalence was calculated for each sample.
The mean and standard deviation was then calculated for the 1000 sample
sets of 10 and 15 observations (PROC MEANS, SAS Institute, Cary, NC).
These results were then compared to real prevalence of copper deficiency
of this population of 33 calves. Real prevalence was calculated by dividing
the number of calves with a liver copper concentrations less than 25
mg/g by the
total number of calves. Liver copper concentrations less than 25 mg/g is the accepted test endpoint for determining
copper deficiency.7 For comparison purposes,
the estimates of the lower confidence limit calculated using the binomial
model were compared with the lower 95% confidence limit calculated using
the Zdistribution. Using the previously described 2 sets of 1000 randomly
selected samples, the real prevalence of copper deficiency was calculated
for each sample of 10 or 15 serum copper determinations using Eq 1.
Thereafter, for each sample the lower 95% confidence limit of calculated
real prevalence was calculated using the Zdistribution: 95% lower confidence
limit = where RP = calculated real prevalence, and n = sample size (either 10 or 15). Thereafter, the mean and standard deviation of the 95% lower confidence limit was calculated for the 1000 randomly selected sample sets containing either 10 or 15 observations.f RESULTS Apparent prevalence based on serum copper concentration was 0.67. Real prevalence based on liver copper concentration was 0.67. The equality of these numbers was coincidental. Results of the model verification trials are as follows. Mean and standard deviation of the 95% lower confidence limit of prevalence calculated using the binomial model of the 10 and 15 sample trials were 0.49 ± 0.24 and 0.64 ± 0.19, respectively. Using the population proportion estimation of the 95% lower confidence limit, the mean and standard deviation of the 10 and 15 sample trials were 0.95 ± 0.14 and 0.99 ± 0.06, respectively. DISCUSSION Application of this model is straightforward. If we sample 5 calves and 4 calves have serum copper concentrations less than 0.45 mg/g, we can confidently assume that herd prevalence of copper deficiency exceeds 40% (Fig. 1). Three positive tests assure us that prevalence is greater than 5%. Two positive tests are consistent with a population with a 0% prevalence of copper deficiency. Cursory appraisal of these results suggests that sample sizes this small should be avoided. As sample size increases, the proportion of positive tests required to reject the hypothesis that prevalence is less than or equal to the hypothesized prevalence becomes smaller. For example, 4 of 5 tests must be positive to reject the hypothesis that prevalence is less than 40%; however, only 5 of 7 or 6 of 9 tests must be positive to reject the same hypothesis (Fig. 1). The estimates of lower confidence limits generated using the binomial model and the Zdistribution differed substantially (Fig. 2). The Zdistribution produced narrow confidence limits; however, these confidence limits are clearly flawed. Lower limits calculated using the Zdistribution exceed real prevalence for both sets analyzed, whether they contain 10 or 15 observations. In contrast, lower confidence limits calculated using the binomial model were less than the real prevalence, and hence, are more plausible and accurate. Comparison of the results of the two methods indicate that use of the traditional method to calculate real prevalence, when the test for evaluation is substantially imperfect, is ill advised. These results reinforce the need for novel approaches to interpret imperfect test results. The binomial model suggested here provides a satisfactory method to interpret imperfect test results. These results highlight some interesting relationships between test results and individual test sensitivities and specificities. In particular, they illustrate the potential for erroneous conclusions when results of imperfect tests are taken at face value. A large proportion of the calves are copperdeficient based upon apparent prevalence. If we were to assume a herd had a real prevalence of copper deficiency of 100%, we could use Eq 2 to calculate the extreme value of apparent prevalence one could expect. In this instance, AP = 1*0.53 + 1*0.89 1 0.89 +1= 0.53. This illustrates that apparent prevalence results based on serum copper concentrations over 53% predict a real prevalence over 100%. In summary, the described
procedure provides a de facto hypothesis test for prevalence. When the
calculated probability is less than 0.025, we have established that
the probability of the observed pattern of test results at the hypothesized
population prevalence is less than 2.5%. In essence, we reject the null
hypothesis that population prevalence is less than the hypothesized
prevalence. In this manner, we are constructing a lower limit confidence
interval for herd prevalence. The described technique
has merit beyond the topic of copper deficiency. Rather than discount
imperfect tests as invalid, we envision using this procedure to develop
confidence limits for population prevalence in those instances in which
test performance, either sensitivity or specificity, of diagnostic tests
is suboptimal. These confidence limits will be appropriate for use
in the development of disease control strategies. REFERENCES 1. Martin SW: Estimating disease prevalence and the interpretation of screening test results. Prev Vet Med 2: 463472, 1984. 2. Daniel WW: Biostatistics: A Foundation for Analysis in the Health Sciences 7^{th} ed. New York: John Wiley & sons, Inc, pp. 176177, 1999. 3. Tessman RK, Lakritz J, Tyler JW, et al: Sensitivity and specificity of serum copper determination for detection of copper deficiency in feeder calves. J Am Vet Med Asso 218:756760, 2001. 4. Martin SW: The evaluation of tests. Can J Comp Med 41:1925, 1977. 5. Moore DS, McCabe GP: Introduction to the Practice of Statistics 2^{nd} ed. New York: W.H. Freeman, pp. 372378, 1993. 6. Pearce SG, Firth EC, Grace ND, et al: Liver biopsy techniques for adult horses and neonatal foals to assess copper status. Aust Vet J 75:194198, 1997. 7. Puls R. Copper. In: Puls R ed. Mineral Levels
in Animal Health 2^{nd} ed. Clearbrook, BC, Canada: Sherpa International,
pp. 82109, 1994.
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Figure 1. Interpretation
of test outcomes for various combinations of number of tests and number
of positive test results. Cells designated by an asterisk (*) denote
the calculated lower limit of copper deficiency prevalence. Figure 2. Results of 2 trials of
randomly generated serum copper concentration sample selection outcomes
for estimation of the lower 95% confidence interval of prevalence of
copper deficiency. The · represents the mean of each trial and the
solid horizontal line represents the standard deviation of the proposed
binomial model. The Ρ represents
the mean of each trial and the solid horizontal line represents the
standard deviation of the traditional method of determination. The vertical
dashed line represents the real prevalence of the population from which
the samples were taken as determined by liver copper concentrations.  
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