Published online September 18, 2006
PEDIATRICS Vol. 118 No. 4 October 2006, pp. e1195-e1202 (doi:10.1542/peds.2005-2925)
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ARTICLE

Sensitivity and Specificity of Various Tests for the Diagnosis of Helicobacter pylori in Egyptian Children

Robert W. Frenck, Jr, MDa, Hanan Mohamed Fathy, MDb, May Sherif, MDc, Zaynab Mohran, PhDc, Hanan El Mohammedy, PhDc, Wagdy Francis, PhDc, David Rockabrand, PhDc, Bahaa Ihab Mounir, MDb, Patrick Rozmajzl, PhDc and Henry F. Frierson, MDd

a Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California
b Department of Pediatrics, Cairo University Faculty of Medicine, Cairo, Egypt
c Enteric Diseases, US Naval Medical Research Unit 3, Cairo, Egypt
d Department of Pathology, University of Virginia Health System, Charlottesville, Virginia


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVES. Many noninvasive methods (using breath, blood, and stool samples) are available to diagnose Helicobacter pylori. However, because the noninvasive tests are proxy measures of the infection, they need validation before use. Factors that may affect test validity include patient age, gender, and geographic location. Because no data were available on the validation of noninvasive tests for the diagnosis of H pylori among children in the Middle East, this study was performed.

METHODS. Children between 2 and 17 years of age evaluated at the Cairo University School of Medicine pediatric gastroenterology clinic who were already scheduled for upper endoscopy were eligible for enrollment in the study. At the time of endoscopy, 3 biopsies were collected and used for rapid urease, histology, and culture, respectively. All children also donated a sample of stool and blood and had a urea breath test performed. Stool and serum samples were tested for the presence of H pylori by using commercially available enzyme-linked immunosorbent assay–based technology. The sensitivity, specificity, and positive and negative predictive values were calculated for each noninvasive test used in the study. Receiver operating curves also were charted to determine optimal cut points for the various tests when used in the current study cohort.

RESULTS. One hundred eight children were enrolled in the study, with 52 children being under 6 years of age. The urea breath test and HpStar (DakoCytomation, Norden, Denmark) stool enzyme-linked immunosorbent assay kit had the highest sensitivity and specificity (sensitivity and specificity: 98 and 89 [urea breath test] and 94 and 81 [HpStar], respectively), whereas the serologic kit had an unacceptably low sensitivity (50%). The sensitivity of neither the urea breath test nor the HpStar tests was affected by subject age, but specificity of the HpStar test, although still high, was significantly lower among children under 6 years. Receiver operating curves found optimal cut points of the urea breath test at 6.2 {delta} over baseline and of the HpStar at 0.25 enzyme-linked immunosorbent assay units.

CONCLUSION. The urea breath test and HpSTAR stool antigen kit are reliable tests for the noninvasive diagnosis of H pylori among children living in the Middle East.

Key Words: Helicobacter • diagnosis • urea breath test • children • Egypt

Abbreviations: NAMRU-3—US Naval Medical Research Unit 3 • UBT—urea breath test • DOB—{delta} over baseline • ELISA—enzyme-linked immunosorbent assay • ROC—receiver operating curve

Helicobacter pylori, identified only 20 years ago, is now known to infect nearly 50% of the world's human population and is responsible for >90% of peptic ulcers.1 Studies also suggest that infection with H pylori in children can increase the risk of diarrheal diseases that may lead to malnutrition and growth faltering.2,3

Diagnosis of H pylori can be made with both invasive and noninvasive tests. Invasive tests, including histology, culture, and rapid urease, require endoscopy to obtain biopsies of the gastric mucosa. Although these tests are highly specific, sensitivity can be affected by the focal distribution of the infection within the stomach.4 In addition, in the developing world, where H pylori is most prevalent, endoscopy may be too resource intensive and laboratory facilities may lack the capability to culture the organism.

Noninvasive tests for the diagnosis of H pylori, which are based on analysis of samples of breath, blood, or stool, have been developed.5 Although these tests are relatively inexpensive and rapid, all are proxy measurements of infection; thus, validation, typically using histology or culture as the gold standard, must be performed initially. Most validation studies have been conducted on adults living in industrialized settings, raising a concern about the applicability of the results to those in the developing world.6–8 An even greater concern is whether test performance in adults can be extrapolated to children. Because no data exist regarding the performance characteristics of invasive or noninvasive tests in children in the Middle East, this study was performed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Patient Population
Children between 2 and 17 years of age who were referred to the pediatric gastroenterology clinic of Cairo University (a tertiary care referral facility in Cairo, Egypt) for an upper endoscopy were eligible to participate. Before endoscopy, parents/guardians were approached in regards to having their child enrolled in the study. Those who expressed interest were explained the study in detail and asked to provide written informed consent to allow their child to be enrolled. Patients who had taken antimicrobial agents, antacids, H2 blockers, a proton-pump inhibitor, or bismuth subsalicylate within 4 weeks before the endoscopy were excluded from the study. Patients were also excluded if they had a past history of infection with H pylori or had a known bleeding disorder or previous endoscopy.

Clinical Evaluation
Before endoscopy, a structured questionnaire was administered to the child's parents to collect information on the presenting symptoms, socioeconomic status, and living conditions of their child, including if there was exposure to farm animals. In addition, height and weight measurements were performed on all subjects. Weights, recorded to the nearest 100 g, were measured while the child was in light clothing. Heights were measured to the nearest centimeter using a length board for children <3 years of age and height sticks for children >3 years of age.

Gastric Biopsy
Three antral biopsies were collected by using an Olympus (Center Valley, PA) ZIF XQ230 fiber-optic endoscope: a rapid urease test was performed on the first biopsy, the second biopsy was sent for histologic evaluation, and the third biopsy was sent for culture.

Blood Collection
A 5-mL sample of blood was collected within 3 days of the endoscopy and before initiation of any therapy against H pylori. After collection, the blood was kept at room temperature for 1 hour, followed by centrifugation at 1500 rpm for 10 minutes. The serum was aliquoted into cryovials and stored at –70°C.

Stool Collection
A sample of stool was collected within 3 days of the endoscopy and before initiation of any therapy against H pylori. After collection, stool samples were stored in a cool box containing ice packs until transferred, within 3 hours of collection, to the US Naval Medical Research Unit 3 (NAMRU-3) in Cairo, Egypt, for processing and testing. After arrival at the NAMRU-3, samples were immediately transferred to cryovials and stored at –70°C until testing was performed.

Urea Breath Test
The Breath Tek urea breath test (UBT) kit (Meretek Diagnostics, Inc, Nashville, TN) was used in this study according to manufacturer recommendations. All samples were collected within 3 days of the endoscopy and before initiation of any therapy against H pylori. After collection of a baseline breath sample, the patient drank 75 mg of [13C]urea dissolved in white grape juice; 30 minutes later, a second breath sample was collected. To minimize background interference from urea-splitting bacteria in the mouth, the juice was consumed through a straw.

All breath samples were collected into a bag with a 1-way valve attached. Whenever possible, the breath sample was collected by the child exhaling directly into the bag. Children unable to exhale directly into the bag had breath samples collected with a pediatric resuscitation mask, which permitted the child to breathe normally as the sample was collected. A fraction of each breath sample was transferred to an evacuated tube and shipped to Meretek Diagnostics, Inc, for analysis. 13C enrichment of respiratory CO2 was measured by automated gas/isotope-ratio mass spectrometry and compared with the reference limestone standard, PeeDee belemnite. The enrichment over baseline was expressed as parts per thousand, and a breath test with ≥5% enrichment over baseline ({delta} over baseline [DOB]) for the test period was considered to be positive for H pylori. In addition, because the amount of urea hydrolyzed is related to the size of the subject, body surface area was calculated and used as another means to define a positive breath test.9 In the case of a discrepant result between the 2 methods, the body surface area method result was used.

Rapid Urease Test
Immediately after collection, 1 biopsy was tested for urease activity by using the "hpfast" test (GI Supply, Camp Hill, PA) according to manufacturer instructions. In brief, the biopsy was placed in an agar gel containing urea and 2 pH dye indicators: bromthymol blue and methyl red. Change of the agar color from yellow to dark green or blue within 24 hours was interpreted as a positive test.

Histopathologic Evaluation
Immediately after collection, 1 biopsy was placed into 10% formalin and stored at room temperature until used for histopathologic examination. A pathologist, blinded to the subject's clinical information, performed the histologic evaluation on all the study biopsies. Tissue was examined for the presence of gastritis, atrophy, and intestinal metaplasia by using the Sydney classification.10,11 Biopsy samples were also stained with hematoxylin and eosin and Giemsa stains to detect H pylori in the tissue.

Bacterial Culture
Immediately after collection, the biopsy sample was placed into 0.1 mL of sterile 0.85% saline in a capped sterile 1.5-mL Eppendorf tube and stored in a cool box until arrival at the NAMRU-3 within 3 hours of collection. At the NAMRU-3 laboratory, the biopsy was placed on ice and homogenized directly in the Eppendorf tube using a sterile pestle. Half of the biopsy homogenate was placed onto a Columbia blood agar plate and the other half onto a Columbia blood agar plate with supplement (trimethoprim, vancomycin, and polymyxin B) and streaked for isolation. The plates were incubated under microaerophilic conditions of 5% O2, 7.5% CO2, 7.5 H2, and 80% N2 using CampyPak microaerophilic system envelopes (Columbia Diagnostics, Springfield, VA) at 37°C for up to 10 days. Plates were checked every other day for growth. On day 5, plates without obvious growth were subcultured onto a Columbia blood agar plate to help promote growth of lightly growing colonies that may have been missed visually. All colonies suggestive of H pylori were tested by Gram-stain, oxidase, catalase, and urease tests to confirm the identification.12

Serum Enzyme-Linked Immunosorbent Assay Testing
Serum samples were tested for the presence of anti–H pylori antibodies by using the HM-CAP enzyme-linked immunosorbent assay (ELISA) kit (EZ-EM, Inc, Westbury, New York) according to manufacturer directions. Samples with an ELISA value of <1.8 ELISA units were considered negative, and samples with an ELISA value of >2.2 ELISA units were considered positive. Samples with values between 1.8 and 2.2 ELISA units were considered indeterminate and were retested. A sample that was still indeterminate after the repeat test was recorded as negative.

Stool Antigen Testing
Stool samples were tested for H pylori by using 2 different antigen-detection kits. Both tests were conducted on the same day and samples were tested in parallel. The amplified IDEIA HpStar test was performed according to manufacturer instructions to detect the presence of H pylori antigen in the stool. Samples having readings ≥0.19 ELISA units were considered positive, and samples with readings <0.19 ELISA units were considered negative. The HpSA kit (Meridian Bioscience, Cincinnati, OH) also was performed according to manufacturer instructions, and samples having an absorbance ≥0.16 ELISA units were considered positive, whereas samples with readings <0.16 ELISA units were considered negative.

Definition of H pylori Infection
The gold standard for classifying a patient as being infected with H pylori was either culturing the organism from the gastric biopsy or, if the culture were negative, having both the rapid urease and histology results be positive. A child was classified as uninfected if all invasive tests gave concordant negative results. Cases with negative culture and discordant results on histology and rapid urease tests were classified as indeterminate and excluded from analysis.

Analytical Plan
Standard methods were used to calculate sensitivity, specificity, and predictive values for positive and negative results of each diagnostic test against the gold standard defined in this study. Previous studies have suggested that the age of the patient may affect the sensitivity and specificity for H pylori testing.13 Therefore, test characteristics were determined on the group as a whole as well as after stratification to children <6 years of age or ≥6 years of age. To compare proportions, the {chi}2 test was used unless the expected cell frequency was <5, in which case Fisher's exact test was used. All statistical testing was 2-tailed, and the level of significance was set at P = .05 unless multiple comparisons were made, in which case the P value was reduced to .001.

Secondary analyses were also performed to determine if sensitivity and specificity of the study tests could be improved. The first iteration classified the noninvasive test on the basis of the results of the UBT combined with the results of a stool antigen kit. The result was classified as positive if both the UBT and the stool antigen test were positive and negative if both tests were negative. Children with discordant UBT and stool antigen test results (ie, UBT-positive and stool antigen–negative) were not included in the analysis. Receiver operating curves (ROCs) also were plotted to determine optimal cut points for the various tests when used in our study cohort (Fig 1).


Figure 1
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  		FIGURE 1 ROC curves of GOLD1 for 4 noninvasive tests in comparison to direct tests for diagnosis of H pylori.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Between June 2002 and February 2003, 108 children were enrolled in the study. Of these, 8 were excluded from final analysis because insufficient samples were available for completion of all the required testing. The invasive tests were able to classify the remaining 100 children as being infected or uninfected with H pylori, and no child had to be excluded from analysis for discordant results. Of the remaining 100 children, 52 were <6 years of age and 48 were ≥6 years of age (Table 1). The most common chief complaints were chronic vomiting (21%), hematemesis (17%), recurrent abdominal pain (13%), and chronic diarrhea (13%). Age stratification (<6 or ≥6 years of age) affected the frequency of a presenting symptom within a group, but the rank order of the symptoms was not affected by stratification. Neither gender of the child nor presenting symptom was statistically more common in either age group of children infected with H pylori as compared with children uninfected with the bacteria.


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  		TABLE 1 Demographics of Study Population

 
Overall, the prevalence of H pylori infection among the study population was 46% (46 of 100 children infected). However, prevalence varied significantly according to age, with 33% (17 of 52) of the children <6 years of age being infected compared with 60% (29 of 48) of the children ≥6 years (P = .005). Of the 46 children classified as being infected with H pylori, the organism was cultured from the biopsy in 44 (96%). Of the 2 H pylori–infected children with negative cultures, one was 5 years old and the other was 6 years old.

Using the above-defined gold standard, the sensitivity, specificity, and predictive values for each of the H pylori indirect diagnostic tests evaluated were calculated and are shown in Table 2. Combining all study children into a single group, the UBT demonstrated the highest sensitivity and specificity (98% and 89%, respectively), followed closely by the HpStar (DakoCytomation, Norden, Denmark) stool antigen-detection test (93% and 88%, respectively). The HpSA stool antigen test and the HM-CAP serum ELISA performed less well than either the UBT or HpStar test.


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  		TABLE 2 Sensitivity, Specificity, and Positive and Negative Predictive Values of Noninvasive Diagnostic Tests for H pylori Infection Among All Children According to Age

 
The large number of study children <6 years of age allowed us to determine if patient age affected test outcomes. Although differences in sensitivity and specificity of the test results were found, the effect of age was not consistent among all the tests (Table 2). For both the UBT and HpStar antigen kit, patient age did not affect test sensitivity. Specificity of the UBT and HpStar tests was lower in the younger children, but the difference was statistically significant between younger and older children only for the HpStar (P = .03). In contrast, sensitivity of the HM-CAP ELISA test was lower in the younger children, whereas both the sensitivity and specificity of the HpSA stool antigen kit were lower in children >6 years of age. However, the variation in test characteristics for the HpSA test between younger and older children was not statistically significant (Table 2).

Subanalyses demonstrated that combination of the UBT and the HpStar test maintained high sensitivity and increased test specificity in the children <6 years of age such that age-related effects were no longer present (Table 2). Combining results of the UBT and HpSA test also resulted in the loss of age-related effects by increasing test specificity in children <6 years of age while increasing sensitivity among the children >6 years of age (Table 2). ROCs indicated that the optimal cut point for the UBT was 6.2 using the DOB method and 15 using the urea-hydrolysis method. For stool assays, the optimal cut point for the DakoCytomation test was 0.25 ELISA units and that for the HpSA test was 0.12 ELISA units. The HM-CAP serum ELISA test was found to be optimized at a cut point of 1.6 ELISA units. Using the new cut points, the sensitivity of the HpSA test rose from 70% to 74% without decreasing test specificity. Similarly, the sensitivity of the HM-CAP test rose from 50% to 59% without decreasing test specificity. The new cut points increased UBT specificity from 89% to 93% without decreasing sensitivity as a result of the better test specificity for children <6 years of age (rising from 86% to 91%).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
This comprehensive study is the first report from Egypt comparing invasive and noninvasive tests to diagnose H pylori in children. In our study, infection with H pylori was common, with an estimated prevalence of 60% among patients >6 years of age. Although this is a high prevalence, we previously reported a prevalence of H pylori infection in Egyptian children of 25% by 3 years of age and rising to 90% by adulthood.14,15 Also, numerous other studies of children in the developing world have reported prevalence of infection exceeding 50% by 3 years of age, indicating that the high percentage of children in our current study infected with H pylori is not unexpected.16,17

The ease of administration and ability to test large numbers of children in a short time have made noninvasive tests the assays of choice for diagnosing H pylori, especially in epidemiologic studies of the infection.18,19 However, noninvasive tests are proxy measurements of infection and need to be validated before routine use. Most validation studies have been performed in adults in industrialized countries.8,20 Validation studies performed in children typically have had small sample sizes, a low percentage of children infected with H pylori, or the UBT, rather than endoscopy with biopsy, used as the gold standard.21–23 This is particularly problematic in children <6 years of age, in whom the sensitivity and specificity of the UBT have not been consistent.24–26

An exception is a recently published multicenter study of European children by Megraud.27 Children from 2 to 17 years of age already scheduled for upper endoscopy were eligible to be enrolled in the H pylori test validation study. After endoscopy, with biopsies taken for culture, histology, and rapid urease tests, children were administered a UBT and had blood, stool, and urine samples collected and tested for H pylori. A total of 473 children were enrolled in the study, and 316 had results available for all 4 noninvasive tests. Of all study children, 40% (191 of 473) were infected with H pylori, with rates ranging from 27% (17 of 48) among children <6 years of age to 45% (120 of 268) in children >6 years of age, quite similar to the findings of our current study. All the noninvasive tests in the Megraud study were highly specific, but the sensitivity of the HpSA and urine ELISA tests were far lower than either the UBT or serologic test. Unfortunately, although the study was designed to evaluate age effects on the sensitivity and specificity of the noninvasive tests, an insufficient number of children <6 years of age infected with H pylori were able to be enrolled into their study, which precluded the ability to meet this important goal.

As with Megraud's study, one of the strengths of our study is that endoscopy with culture, histology, and rapid urease testing was performed on all the study patients, which allowed for definitive diagnosis of H pylori and provided a strong gold standard against which to validate the noninvasive tests in children. In addition, our study included 52 children <6 years of age with a 33% prevalence of infection with H pylori, which allowed stratification of results by patient age.

In this study, we demonstrated that noninvasive tests, particularly the UBT and the HpStar stool antigen test, are highly sensitive and specific for the diagnosis of H pylori in children. We found that the sensitivity of the UBT was not affected by the age of the patient but specificity was lower, although not statistically, in younger children (86% vs 95%, children <6 vs ≥6 years, respectively; P = .44). A possible reason for the lower specificity in younger children is the presence of urea-splitting bacteria in the mouth, particularly Streptococcus salivarius, which may have released the 13C from the urea.28 To minimize this possibility, children drank the grape juice containing urea through a straw, but it still may not have totally prevented exposure of the urea to mouth flora.

Consistent with other studies from the developing world, we found that serology did not perform as well as other noninvasive tests used in this project.19,21,29 The serologic kit used in this study (HM-CAP) was selected on the basis of the high sensitivity and specificity of the test in children.30 However, it seems that test characteristics of the HM-CAP kit may not be consistent throughout the world, possibly because of H pylori circulating in the United States varying from those in other parts of the world.

Stool antigen tests to diagnose H pylori also seem to vary in sensitivity and specificity. Similar to the results of the Megraud study, the HpSA test had a sensitivity of 70%, but unlike the findings of Megraud, the specificity of the test in our study was only 77%. In addition, an unexpected finding was that the HpSA test performed better in children <6 years of age compared with older children, largely because of the number of false-negative results in the older children. The HpStar stool antigen kit performed very well in all age groups tested, although the specificity was slightly but significantly lower in the younger children. The results of our study compare favorably with a study by Sykora et al,31 in which the HpStar test had a sensitivity of 96.1% and specificity of 98.5% along with positive and negative predicting values of 96.1% and 98.5%, respectively.31 The difference in results obtained with the HpSA test compared with the HpStar test is not certain but may be a result of the use of polyclonal antibodies in the HpSA assay, whereas the HpStar test uses a monoclonal antibody for antigen capture.32

We also evaluated the possibility of improving the sensitivity and specificity of the noninvasive tests to diagnose H pylori, particularly in children <6 years of age. As a first subanalysis, the results of 2 noninvasive tests were combined, with the set being considered positive if both noninvasive tests were positive and negative if both tests were negative. With this technique, specificity and sensitivity, along with positive and negative predictive values, all exceeded 90% when the UBT was combined with one of the stool antigen tests, and age effects were no longer present. A possible clinical utility of the method would be for confirmation of a positive test in a younger child. Using such a strategy, positive results, even in children <6 years of age, would strongly indicate that the child was infected with H pylori. The only downside of this method was a relatively high percentage of discordant results (10% when the UBT and HpStar test were combined and 33% for combination of the UBT and HpSA test). The high negative predictive value of the UBT or stool antigen test seems to preclude the need to confirm a negative finding with a second noninvasive test.

A second subanalysis developed ROCs to determine the optimal cut point for the various noninvasive tests used in the study. Our findings were similar to Megraud's in that test sensitivity, especially for the UBT in children <6 years of age, could be increased without significantly altering test specificity. The optimized cut point of 6.2 DOB for the UBT in our study matches well with that reported by Thomas et al,33 who found an optimal DOB of 5.6 in a study of H pylori in children living in the Gambia, far higher than the level of 2.5 DOB that is routinely used as a cutoff level for a positive UBT in adults.34

A limitation of our study is that all children were recruited from a gastroenterology clinic in which they were undergoing evaluation of various gastrointestinal complaints. However, ethical considerations preclude performing endoscopy on healthy children. Dilution of the stool among the children with a chief complaint of diarrhea possibly could have decreased the accuracy of the stool antigen tests. However, a laboratory study testing this possibility found that even after a 50-fold dilution, only 1 of 9 stool samples from children infected with H pylori changed from positive to negative by H pylori stool antigen testing.35 Therefore, it is unlikely that the stool antigen testing results in this study were affected by the children having diarrhea as their chief complaint. Another possible study limitation is the collection and storage of samples at Cairo University before transfer to the NAMRU-3, in which laboratory testing was performed. However, the samples were stored at Cairo University for a maximum of 3 hours before cross-town transport. Once the samples arrived at the NAMRU-3, they were processed immediately, with the blood and stool stored at –70°C until the day of testing. The only test result that may have been affected by transport and storage was the HpSA test, which had a large number of false-negative tests. Additional studies would need to be conducted to test this possibility.


   CONCLUSIONS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
The UBT and the HpStar stool antigen test were highly sensitive and specific noninvasive tests for the diagnosis of H pylori in Egyptian children. Both tests were easy to perform and are readily available. The specificity of the UBT was slightly lower in the younger children but still provided important clinical information. The ability to obtain accurate results rapidly without the need for sophisticated laboratory equipment may make the HpStar test the noninvasive test of choice in resource-poor settings. The sensitivity of the serologic test used in the current study was unacceptably low and would need additional evaluation before it could be recommended for use outside of the United States.


   ACKNOWLEDGMENTS
 
Drs Shannon Putnam and John Sanders provided thoughtful review and critique of the manuscript. We appreciate the assistance of Stanley J. Konopka, PhD (Meretek Diagnostics, Inc), in the analysis of breath samples. Finally, we acknowledge the invaluable mentoring that the late Peter D. Klein, PhD, provided to us on the conduct of the study.


   FOOTNOTES
 
Accepted May 23, 2006.

Address correspondence to Robert W. Frenck Jr, MD, Harbor-UCLA Medical Center, 1124 Carson St, Torrance, CA 90502. E-mail: rfrenck{at}uclacvr.labiomed.org

The opinions and assertions herein should not be construed as official or representing the views of the Department of the Navy, the Department of Defense, or the US government. This is a US government work; there are no restrictions on its use.

The authors have indicated they have no financial relationships relevant to this article to disclose.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics




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