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Screening of half a million Swedish newborn infants for congenital adrenal hyperplasia
Screening, I(l992) 159-166 0 1992 Elsevier Science Publishers B.V. All rights reserved 0925-6164/92/$5.00 SCREEN 00027
Screening of half a million Swedish newborn infants for congenital adrenal hyperplasia Agne Larsson, Astrid ThilCn, Lars Hagenfeldt, Ulrika von Diibeln and Claes Guthenberg Department of Pediatrics, Uppsala University, Uppsala. Sweden and the PKU Seetion, National Bacteriological Laboratory, Stockholm, Sweden
(Accepted 6 March 1992)
Using filter paper blood samples, 557,000 newborn infants were screened for congenital adrenal hyperplasia (CAH). 17-Hydroxyprogesterone was determined by radioimmunoassay without organic solvent extraction. By the use of gestational age-related cut-off limits the false positive rate could be decreased to less than 0.03% (1 : 4000). Two thirds of the falsepositive cases were preterm infants, with a mean gestational age of 28 weeks (range 24-36 weeks). The prevalence of CAH in the screened population was 1: 11,600, which does not differ from the figure obtained before the start of screening. The sensitivity of the screening test was 0.92 and the predictive value of a positive screening test was 0.24. The screening result was available at the age of 11 days (median). By screening it was possible to avoid serious salt loss after the age of 2 weeks. Screening resulted in earlier diagnosis in at least 50% of all cases of CAH. It also led to earlier correct gender assignment in virilizeld girls. The cost of screening was approximately 2.6 USS per infant (or 32,000 US$ per CAH patient). We conclude that the benefits of screening are significant, and suggest that CAH screening should be included in the routine neonatal screening program in Sweden. Key words. Neonatal screening; Congenital adrenal hyperplasia; 21-Hydroxylase deficiency;
Adrenogenital syndrome; 17-Hydroxyprogesterone
Introduction The diagnosis of congenital adrenal hyperplasia (CAH) is often difficult to establish on the basis of clinical symptoms [2,12]. The prevalence of classical CAH in Sweden was 1 : 11,500 prior to screening and in 66% of the patients treatment was started later than at the age of 2 weeks [12]. Laboratory screening based on radioCorrespon(dence to: Dr. Agne Larsson, Department of Pediatrics, Uppsala University Children’s Hospital, S-751 85 IJppsala, Sweden.
160
immunoassay of 17-hydroxyprogesterone (17-OHP) in filter paper blood samples was first described by Pang et al. [6] and has subsequently been tested by others [1,3,6,9,10,11,13]. The need for improved diagnostic facilities for patients with CAH has been emphasized [7]. We have tested nationwide screening for CAH since 1986 in order to evaluate its benefits. Material and Methods Filter paper blood spots were used. They were collected from all newborn infants on days 3-5 after birth and the gestational age of the child was recorded. 17-Hydroxyprogesterone was determined by radioimmunoassay (MILAB, Malmii, Sweden) as described earlier, [6] i.e., without extraction with organic solvent, in a single 3 mm disk. When elevated 17-OHP was found, the sample was reanalyzed after ether extraction. The initial recall level was set at 200 nmol/l plasma (100 nmol/l blood) based on a study of stored samples from positive cases [6]. After an initial trial period, recall levels adjusted for gestational age (Table 1) were introduced to avoid unacceptable high recall rate among preterm infants [6]. During the period covered by the present report, the recall level for term infants was lowered from 200 to 150 nmol/l plasma (after 300,000 screened infants). This change had no effect on the rate of false-positive results. As soon as a screening test was classified as positive, the pediatrician in charge of the patient was informed by telephone. The cost per child was 15 SEK (approximately 2.6 US$), expressed in the 1991 value, including reagents, equipment and salaries. Criteria for classification of a positive screening test as true were elevated serum 17-OHP according to the reference range used by the local laboratory, a typical urine steroid hormone metabolite pattern as evaluated by gas chromatography/mass spectrometry [6] and one or more of the following signs and symptoms of CAH: virilization, hyperpigmentation, failure to thrive, hypotonia, hyponatremia/ hyperkalemia. All infants with positive screening tests have undergone continuous clinical followup. During the first 3 years of screening (1986- 1988) information about such neonates TABLE 1 Cut-off limits of 17-hydroxyprogesterone (17-OHP) in CAH screening Gestational age (weeks)
17-OHP (nmol/l plasma)
Action taken
<32
> 600 200-599
33-36
3 400 200-399
>37
2150
recall directly reassay of 17-OHP after ether extraction; recall if a200 nmol/l recall directly reassay of 17-OHP after ether extraction; recall if > 200 nmol/l recall directly
161
was obtained by telephone interviews with the pediatrician in charge of the patient and through copies of the hospital records. In order to get more detailed information the follow-up was changed in 1989-1990: every child who had a positive screening test or who was suspected to have CAH on the basis of clinical signs and symptoms (before the screening result was available or when the screening test was negative) was imm’ediately reported to one of the authors (AT). The pediatrician in charge of the patient was interviewed by telephone as soon as possible. Questionnaires were used for the first follow-up of a positive case and for the subsequent clinical examinations in true-positive cases. At the time of diagnosis information was obtained about heredity and the pregnancy, delivery and neonatal period. If the CAH diagnosis was confirmed, the additional follow-up program involved examination of the child every 3 months during the first year of life and every 6 months thereafter. Special attention was paid to physical and psychosomatic development, virilization, changes in therapy, intercurrent diseases, surgery and changes in the social situation of the family. Every child with CAH was examined by one of the authors (AT) together with the pediatrician in charge of the patient as soon as possible after the alert had been given from the screening laboratory, usually within 1 month after the diagnosis had been established.
Results
The overall result of the screening from 1986 through 1990 is shown in Table 2. Over half a million infants were screened. The predictive value of a positive test was 0.24. The sensitivity was 0.92 and the specificity was >0.9999. On average, the screening sample was collected on day 4 (median) and reached the screening laboratory by mail on day 7, and the 17-OHP result was available on day 11 (range: 7-19 days). In patients detected by screening the result was available on day 13, whereas in patients in whom CAH was suspected before the screening outcome was ready the result was obtained on day 9. This difference was due to the fact that the blood sample was collected earlier if CAH was suspected on clinical grounds. Often the clinician informed the screening laboratory that the child was suspected to have CAH and the screening analysis was given high priority.
TABLE 2 Overall rewlt of CAH screening from 1986 through 1990 Screened infants Positive te,sts False-positive True-positive False-negative tests
557,410 183 139 44 (24 F, 20 M) 4 (4 F)
162
True-positive cases
Forty-nine (29 F, 20 M) Swedish children born in 1986 through 1990 were given a diagnosis of CAH up to December 1991. Out of these, 44 (24 F, 20 M) patients were identified in the screened population as true-positive cases (Table 2). Four girls were classified as false-negative and one girl had not been screened since she was born abroad. The prevalence of CAH was 1 : 11,600 in the screened population. The median gestational age of the true-positive cases was 40 weeks (range: 36-42 weeks). In the initial screening blood sample the median level of 17-OHP before ether extraction was 674 nmol/l plasma (range: 1455> 1000) and after ether extraction 469 nmol/l plasma (range: 42- > 1000). The follow-up serum samples were analyzed at local laboratories using different commercial radioimmunoassays for 17-OHP and the results were evaluated against the reference range established in the local laboratories for children of the corresponding age. The median serum 17-OHP was 400 nmol/l (range: 36-3000). In a reference range for children 2 to 7 days of age was estimated by Hughes et al. [5], the mean plasma concentration of 17-OHP was found to be 3.5 nmol/l in normal infants and up to 30 nmol/l in some stressed infants. Clinical findings in the 44 infants with true positive tests are summarized in Table 3. In this group 52% were detected by screening. The nine asymptomatic patients (2 F, 7 M) had serum sodium values of 2 130 mmol/l, no virilization and no general symptoms such as poor weight gain or weight loss of more than 10% of the weight at birth, feeding problems, tiredness or obvious improvement in their general condition when therapy was started. In many cases the clinical symptoms were mild and not always mentioned in the standard hospital records or in the questionnaires, but were revealed by examination of the child performed by one of the authors (AT) or by interviews with the parents and the pediatrician in charge. TABLE 3 True-positive cases in the CAH screening
Number of patients Asymptomatic* Virilization only Virilization and salt loss Salt loss only Macrogenitosomia and/or hyperpigmentation Incorrect gender assignment Sibling with CAH
All cases suspected
Diagnosed by screening
Diagnosis before screening result
44 (24 F, 20 M) 9 (2 F, 7 M)
23 8 3 2 7 3
21 (17 F, 4 M)
11 (F) 10 (F) 10 (W 4 (M)
(7 F, 16 M) (2 F, 6 M) (F) (F) (M) (M)
1 W 8 (F) 8 F) 3 (M)
1 CM)
5 (F)
1 (F)
4 (F)
4 (2 F, 2 M)
1 CM)
3 (2 F, 1 M)
*Serum sodium 2 130 mmol/l, no general symptoms and no virilization.
163
Hyponatremia, i.e., serum sodium below 130 mmol/l, occurred in 20 of the 44 CAH patients (Table 3). Nine of these 20 patients were detected by screening and in the other 11, CAH was suspected before the alert was given from the screening laboratory. Severe general symptoms and hyponatremia requiring parenteral therapy with sodium and hydrocortisone occurred in five patients (1 F, 4 M). Twelve children (7 F, 5 14) had subnormal serum sodium values (130-133 mmol/l), mostly in combination with mild general symptoms. Hyperkalemia, i.e., serum potassium above 7 mmol/l, occurred in 16 (9 F, 7 M) patients. As the serum samples were mostly collected by a capillary technique, the hyperkalemia was usually regarded as an artifact due to hemolysis. It is noteworthy, however, that patients with hyperkalemia often showed general clinical symptoms, even if their serum sodium was in the range of 130-133 mmol/l. Five patients with salt loss manifested hyperkalemia between 8.510 mmol/l. Virilization was found in 21 of the 24 girls with CAH. Two non-virilized girls were diagnosed by screening and they did not have any additional symptoms. Their diagnoses were confirmed by the urine steroid hormone profile. A third girl who was not virilized at birth had been diagnosed prenatally because she had an older sibling with CAH and the mother had been treated with dexamethasone since week 6 of pre,gnancy. Five clearly virilized girls were detected by screening. One of these girls was mistaken for a boy with hypospadia and cryptorchidism. Incorrect gender assignment was made in five girls with CAH, who were mistaken for boys, with hypospadia and cryptorchidism for a period of 7-13 days. In one of the girls a consultant pediatric surgeon suspected the presence of a testis in the right inguen. This girl was suspected of having CAH at the age of 10 days when she was admitted to hospital because of a urinary tract infection, and was then examined more thoroughly. The screening result was available on day 12. One of the girls with incorrect gender assignment was diagnosed by the screening. In four girls in whom CAH was suspected before the screening result was available, gender assignment was a problem for l-6 days (median 3.5). Treatment with glucocorticoid and mineralocorticoid hormone was usually started at the time of the definite diagnosis. The median age at the start of treatment was 14.5 days (range: 5-36). Only two boys out of the 44 patients classified as truepositive were not receiving replacement therapy at the ages of 3.5 and 4 years, respectively. They have remained free of symptoms, but the older boy had a bone age of -I- 3.5 SD. Their plasma concentrations of 17-OHP at screening were 243 and 230 nmol/l. Their CAH diagnosis was confirmed by urine steroid hormone analysis and elevated serum 17-OHP (230 and 113 nmol/l).
False-positive cases
With use of the gestational age-related cut-off limits the overall false-positive rate was 0.03% (1 : 4000) (Table 2). In spite of the gestational age-related limits, 67% of all false-positive cases were born preterm. Their mean gestational age was 28 weeks (range: 24-36). The majority of the false-positive infants were hospitalized in neonatal wards at the time of the alert.
164
False-negative cases
Four missed cases of CAH were identified in the screened population. Case I. This girl was born at term. Her screening sample contained ~40 nmol
17-OHP/l plasma. She was diagnosed at the age of 2.5 years because of growth acceleration and clitoris enlargement (present from birth according to the mother). She had had three episodes of fever and vomiting, requiring intravenous rehydration on one occasion. No abnormal serum electrolyte levels were recorded. The CAH diagnosis was confirmed by the urine steroid hormone profile. She was considered to have simple virilizing CAH. Case ZZ. This girl was born at term. She had clitoris enlargement and labial fusion. Serum Na was 133-137 and K 7.4 mmol/l. The concentration of 17-OHP in her screening sample on day 4 was 118 nmol/l plasma. A second filter paper sample on day 11 gave a value of 394 nmol of 17-OHP/l plasma (323 nmol/l after ether extraction) and a serum sample on the same day of 249 nmol/l serum. The CAH diagnosis was confirmed by the urine steroid hormone pattern and treatment was started on day 19. Case ZZZ. This girl had an elder sister with late-diagnosed mild CAH. This information was unfortunately not conveyed to the screening laboratory. The girl was born at term and was not virilized. Her neonatal screening sample was negative, i.e., the 17-OHP concentration was 152 nmol/l plasma (cut-off limit at that time 200 nmol/l plasma). After the age of 1.5 years she developed clitoris enlargement, pubarche and accelerated skeletal maturation (bone age was + 3.5 SD at 2 years of age). Her basal serum 17-OHP at the age of 2 years was 800 nmol/l and after ACTH stimulation it increased markedly to 1500 nmol/l with a small serum cortisol increase (from 164 to 260 nmol/l). Treatment was begun at the age of 2.6 years. Case IV. This girl was born at term. She had clitoris enlargement and urogenital sinus and CAH was therefore suspected. Serum electrolytes were normal. Her screening sample on day 5 contained 125 nmol 17-OHP/l plasma (cut off limit 150). Therapy was started on day 10 after an analysis performed at the local laboratory had shown a serum 17-OHP concentration of 92 nmol/l (reference range: < 4 nmol/l). Unscreened CAH patient
One Swedish child with CAH was not screened because she was born abroad after 28 weeks of gestation. She was initially considered to be a boy with hypospadia and cryptorchidism. The child developed significant hyponatremia (lowest serum sodium 126 mmol/l). The CAH diagnosis was established at the age of 25 days. The child’s gender was then corrected and hormone supplementation was initiated.
Discussion
The present study comprised more than 0.5 million newborn infants screened for CAH. The goal of the screening was to detect patients with CAH before they were diagnosed on grounds of serious symptoms. More than 90% of all known CAH
165
patients were diagnosed before the age of 2 weeks. This is considerably earlier than was the case before screening was started [12]. The screening analysis led to earlier diagnosis in 52% of the CAH patients, whereas in the remaining 48% - mostly girls - the diagnosis had already been made or was highly suspected at the time when the screening result became available. The median age at which the CAH diagnosis in the screened population was established and treatment with cortisone was begun was 12 days, compared with 30 days before screening [12]. By screening it was possible to avoid serious salt-loss crises after the age of 2 weeks. Incorrect gender assignment is a problem in girls with CAH [12]. There was considerable delay in correct gender assignment before screening, but not after. Additional benefits of CAH screening are that virilization, growth acceleration and prernature pubarche are decreased. An imeresting observation was that the earlier mentioned nonvirilized girl with CAH whose mother was treated with dexamethasone 1.5-1.75 mg daily from the sixth week of gestation to the end of pregnancy nevertheless had elevated 17-OHP in her screening sample collected 3 days after birth; she was born at term and had more than 700 nmol of 17-OHP/l of plasma before and 574 nmol/l after extraction of the sa.mple with ether. A similar case was reported earlier [3]. Congenital adrenal hyperplasia is a very heterogeneous condition [2,14]. Multiple mutant 21-hydroxylase alleles have now been identified. It is likely that there is a continuum of deficiencies rather than only two categories of CAH as assumed previously, i.e., classical CAH and the late-onset or cryptic type. It is therefore not surprising that a screening system will have occasional false-negative test results. We encountered four such cases. It is also possible that patients with mild CAH, missed by screening, will be identified in the future. The false-positive group consisted mainly of preterm infants, in spite of the use of gestational age-related cut-off limits. The infants were usually still in neonatal wards at the time of the alert and a second follow-up sample could easily be obtained. We conclude that the benefits of CAH screening are significant and recommend adding CAH screening to the neonatal metabolic screening program in Sweden, which would then include PKU, galactosemia, congenital hypothyroidism and CAH.
Acknowledgement This stu’dy was supported by grants from the Swedish Medical Research Council (4792).
References 1 Cacciari E, Balsam0 A, Cassio A et al. Neonatal screening for congenital adrenal hyperplasia using a microfilter paper method for 17-a-hydroxyprogesterone radioimmunoassay. Horm Res 1982;16:4-9. 2 Cutler Jr GB, Laue L. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. New Engl J Med 1990;323:1806-1813.
3 David M, Forest MG. Prenatal treatment of congenital adrenal hyperplasia resulting from 21hydroxylase deficiency. Pediatrics 1984; 105:799-803. 4 Farriaux JP, Dhondt JL, Dorche C et al. Depistage neonatal de l’hyperplasie congenitale des surrenales par deficit en 2l-hydroxylase. Realites et perspectives. Arch Fr Pediatr 1987;44:739-747. 5 Hughes IA, Riad-Fahmy D, Griffiths K. Plasma 170H-progesterone concentrations in newborn infants. Arch Dis Childhood 1979;54:347-349. 6 Larsson A, Hagenfeldt L, von Dijbeln U et al. Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots. Horm Res 1988;30:235-240. 7 Lebovitz RM, Pauli RM, Laxova R. Delayed diagnosis in congenital adrenal hyperplasia: need for newborn screening. Am J Dis Child 1984;138:571-573. 8 Pang S, Hotchkiss J, Drash AL et al. Microfilter paper method for 17a-hydroxyprogesterone radioimmunoassay: its application for rapid screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1987;45: 1003-1008. 9 Pang S, Wallace MA, Hofman L et al. Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 2l-hydroxylase deficiency. Pediatrics 1988;81:866-874. IO Piazzi S, Capelli M, Paolini M et al. Neonatal screening for 21-hydroxylase deficiency: a microfilter paper method for l7a-hydroxyprogesterone assay. J Endocrinol Invest 1982;5:87-90. I I Shimozawa K, Saisho S, Saito N et al. A neonatal mass-screening for congenital adrenal hyperplasia in Japan. Acta Endocrinol 1984;107:513-518. I2 Thiltn A, Larsson A. Congenital adrenal hyperplasia in Sweden 1969-1986. Acta Paediatr Stand 1990;79:168-175, 13 Wallace AM, Beastall GH, Cook B et al. Neonatal screening for congenital adrenal hyperplasia: a programme based on a novel direct radioimmunoassay for l7-hydroxyprogesterone in blood spots. J Endocrinol 1986;108:299-308. I4 White PC, New MI, DuPont B. Congenital adrenal hyperplasia I and II. New Engl J Med 1987;316:1519-24 & 1580-1586.
Screening of half a million Swedish newborn infants for congenital adrenal hyperplasia Agne Larsson, Astrid ThilCn, Lars Hagenfeldt, Ulrika von Diibeln and Claes Guthenberg Department of Pediatrics, Uppsala University, Uppsala. Sweden and the PKU Seetion, National Bacteriological Laboratory, Stockholm, Sweden
(Accepted 6 March 1992)
Using filter paper blood samples, 557,000 newborn infants were screened for congenital adrenal hyperplasia (CAH). 17-Hydroxyprogesterone was determined by radioimmunoassay without organic solvent extraction. By the use of gestational age-related cut-off limits the false positive rate could be decreased to less than 0.03% (1 : 4000). Two thirds of the falsepositive cases were preterm infants, with a mean gestational age of 28 weeks (range 24-36 weeks). The prevalence of CAH in the screened population was 1: 11,600, which does not differ from the figure obtained before the start of screening. The sensitivity of the screening test was 0.92 and the predictive value of a positive screening test was 0.24. The screening result was available at the age of 11 days (median). By screening it was possible to avoid serious salt loss after the age of 2 weeks. Screening resulted in earlier diagnosis in at least 50% of all cases of CAH. It also led to earlier correct gender assignment in virilizeld girls. The cost of screening was approximately 2.6 USS per infant (or 32,000 US$ per CAH patient). We conclude that the benefits of screening are significant, and suggest that CAH screening should be included in the routine neonatal screening program in Sweden. Key words. Neonatal screening; Congenital adrenal hyperplasia; 21-Hydroxylase deficiency;
Adrenogenital syndrome; 17-Hydroxyprogesterone
Introduction The diagnosis of congenital adrenal hyperplasia (CAH) is often difficult to establish on the basis of clinical symptoms [2,12]. The prevalence of classical CAH in Sweden was 1 : 11,500 prior to screening and in 66% of the patients treatment was started later than at the age of 2 weeks [12]. Laboratory screening based on radioCorrespon(dence to: Dr. Agne Larsson, Department of Pediatrics, Uppsala University Children’s Hospital, S-751 85 IJppsala, Sweden.
160
immunoassay of 17-hydroxyprogesterone (17-OHP) in filter paper blood samples was first described by Pang et al. [6] and has subsequently been tested by others [1,3,6,9,10,11,13]. The need for improved diagnostic facilities for patients with CAH has been emphasized [7]. We have tested nationwide screening for CAH since 1986 in order to evaluate its benefits. Material and Methods Filter paper blood spots were used. They were collected from all newborn infants on days 3-5 after birth and the gestational age of the child was recorded. 17-Hydroxyprogesterone was determined by radioimmunoassay (MILAB, Malmii, Sweden) as described earlier, [6] i.e., without extraction with organic solvent, in a single 3 mm disk. When elevated 17-OHP was found, the sample was reanalyzed after ether extraction. The initial recall level was set at 200 nmol/l plasma (100 nmol/l blood) based on a study of stored samples from positive cases [6]. After an initial trial period, recall levels adjusted for gestational age (Table 1) were introduced to avoid unacceptable high recall rate among preterm infants [6]. During the period covered by the present report, the recall level for term infants was lowered from 200 to 150 nmol/l plasma (after 300,000 screened infants). This change had no effect on the rate of false-positive results. As soon as a screening test was classified as positive, the pediatrician in charge of the patient was informed by telephone. The cost per child was 15 SEK (approximately 2.6 US$), expressed in the 1991 value, including reagents, equipment and salaries. Criteria for classification of a positive screening test as true were elevated serum 17-OHP according to the reference range used by the local laboratory, a typical urine steroid hormone metabolite pattern as evaluated by gas chromatography/mass spectrometry [6] and one or more of the following signs and symptoms of CAH: virilization, hyperpigmentation, failure to thrive, hypotonia, hyponatremia/ hyperkalemia. All infants with positive screening tests have undergone continuous clinical followup. During the first 3 years of screening (1986- 1988) information about such neonates TABLE 1 Cut-off limits of 17-hydroxyprogesterone (17-OHP) in CAH screening Gestational age (weeks)
17-OHP (nmol/l plasma)
Action taken
<32
> 600 200-599
33-36
3 400 200-399
>37
2150
recall directly reassay of 17-OHP after ether extraction; recall if a200 nmol/l recall directly reassay of 17-OHP after ether extraction; recall if > 200 nmol/l recall directly
161
was obtained by telephone interviews with the pediatrician in charge of the patient and through copies of the hospital records. In order to get more detailed information the follow-up was changed in 1989-1990: every child who had a positive screening test or who was suspected to have CAH on the basis of clinical signs and symptoms (before the screening result was available or when the screening test was negative) was imm’ediately reported to one of the authors (AT). The pediatrician in charge of the patient was interviewed by telephone as soon as possible. Questionnaires were used for the first follow-up of a positive case and for the subsequent clinical examinations in true-positive cases. At the time of diagnosis information was obtained about heredity and the pregnancy, delivery and neonatal period. If the CAH diagnosis was confirmed, the additional follow-up program involved examination of the child every 3 months during the first year of life and every 6 months thereafter. Special attention was paid to physical and psychosomatic development, virilization, changes in therapy, intercurrent diseases, surgery and changes in the social situation of the family. Every child with CAH was examined by one of the authors (AT) together with the pediatrician in charge of the patient as soon as possible after the alert had been given from the screening laboratory, usually within 1 month after the diagnosis had been established.
Results
The overall result of the screening from 1986 through 1990 is shown in Table 2. Over half a million infants were screened. The predictive value of a positive test was 0.24. The sensitivity was 0.92 and the specificity was >0.9999. On average, the screening sample was collected on day 4 (median) and reached the screening laboratory by mail on day 7, and the 17-OHP result was available on day 11 (range: 7-19 days). In patients detected by screening the result was available on day 13, whereas in patients in whom CAH was suspected before the screening outcome was ready the result was obtained on day 9. This difference was due to the fact that the blood sample was collected earlier if CAH was suspected on clinical grounds. Often the clinician informed the screening laboratory that the child was suspected to have CAH and the screening analysis was given high priority.
TABLE 2 Overall rewlt of CAH screening from 1986 through 1990 Screened infants Positive te,sts False-positive True-positive False-negative tests
557,410 183 139 44 (24 F, 20 M) 4 (4 F)
162
True-positive cases
Forty-nine (29 F, 20 M) Swedish children born in 1986 through 1990 were given a diagnosis of CAH up to December 1991. Out of these, 44 (24 F, 20 M) patients were identified in the screened population as true-positive cases (Table 2). Four girls were classified as false-negative and one girl had not been screened since she was born abroad. The prevalence of CAH was 1 : 11,600 in the screened population. The median gestational age of the true-positive cases was 40 weeks (range: 36-42 weeks). In the initial screening blood sample the median level of 17-OHP before ether extraction was 674 nmol/l plasma (range: 1455> 1000) and after ether extraction 469 nmol/l plasma (range: 42- > 1000). The follow-up serum samples were analyzed at local laboratories using different commercial radioimmunoassays for 17-OHP and the results were evaluated against the reference range established in the local laboratories for children of the corresponding age. The median serum 17-OHP was 400 nmol/l (range: 36-3000). In a reference range for children 2 to 7 days of age was estimated by Hughes et al. [5], the mean plasma concentration of 17-OHP was found to be 3.5 nmol/l in normal infants and up to 30 nmol/l in some stressed infants. Clinical findings in the 44 infants with true positive tests are summarized in Table 3. In this group 52% were detected by screening. The nine asymptomatic patients (2 F, 7 M) had serum sodium values of 2 130 mmol/l, no virilization and no general symptoms such as poor weight gain or weight loss of more than 10% of the weight at birth, feeding problems, tiredness or obvious improvement in their general condition when therapy was started. In many cases the clinical symptoms were mild and not always mentioned in the standard hospital records or in the questionnaires, but were revealed by examination of the child performed by one of the authors (AT) or by interviews with the parents and the pediatrician in charge. TABLE 3 True-positive cases in the CAH screening
Number of patients Asymptomatic* Virilization only Virilization and salt loss Salt loss only Macrogenitosomia and/or hyperpigmentation Incorrect gender assignment Sibling with CAH
All cases suspected
Diagnosed by screening
Diagnosis before screening result
44 (24 F, 20 M) 9 (2 F, 7 M)
23 8 3 2 7 3
21 (17 F, 4 M)
11 (F) 10 (F) 10 (W 4 (M)
(7 F, 16 M) (2 F, 6 M) (F) (F) (M) (M)
1 W 8 (F) 8 F) 3 (M)
1 CM)
5 (F)
1 (F)
4 (F)
4 (2 F, 2 M)
1 CM)
3 (2 F, 1 M)
*Serum sodium 2 130 mmol/l, no general symptoms and no virilization.
163
Hyponatremia, i.e., serum sodium below 130 mmol/l, occurred in 20 of the 44 CAH patients (Table 3). Nine of these 20 patients were detected by screening and in the other 11, CAH was suspected before the alert was given from the screening laboratory. Severe general symptoms and hyponatremia requiring parenteral therapy with sodium and hydrocortisone occurred in five patients (1 F, 4 M). Twelve children (7 F, 5 14) had subnormal serum sodium values (130-133 mmol/l), mostly in combination with mild general symptoms. Hyperkalemia, i.e., serum potassium above 7 mmol/l, occurred in 16 (9 F, 7 M) patients. As the serum samples were mostly collected by a capillary technique, the hyperkalemia was usually regarded as an artifact due to hemolysis. It is noteworthy, however, that patients with hyperkalemia often showed general clinical symptoms, even if their serum sodium was in the range of 130-133 mmol/l. Five patients with salt loss manifested hyperkalemia between 8.510 mmol/l. Virilization was found in 21 of the 24 girls with CAH. Two non-virilized girls were diagnosed by screening and they did not have any additional symptoms. Their diagnoses were confirmed by the urine steroid hormone profile. A third girl who was not virilized at birth had been diagnosed prenatally because she had an older sibling with CAH and the mother had been treated with dexamethasone since week 6 of pre,gnancy. Five clearly virilized girls were detected by screening. One of these girls was mistaken for a boy with hypospadia and cryptorchidism. Incorrect gender assignment was made in five girls with CAH, who were mistaken for boys, with hypospadia and cryptorchidism for a period of 7-13 days. In one of the girls a consultant pediatric surgeon suspected the presence of a testis in the right inguen. This girl was suspected of having CAH at the age of 10 days when she was admitted to hospital because of a urinary tract infection, and was then examined more thoroughly. The screening result was available on day 12. One of the girls with incorrect gender assignment was diagnosed by the screening. In four girls in whom CAH was suspected before the screening result was available, gender assignment was a problem for l-6 days (median 3.5). Treatment with glucocorticoid and mineralocorticoid hormone was usually started at the time of the definite diagnosis. The median age at the start of treatment was 14.5 days (range: 5-36). Only two boys out of the 44 patients classified as truepositive were not receiving replacement therapy at the ages of 3.5 and 4 years, respectively. They have remained free of symptoms, but the older boy had a bone age of -I- 3.5 SD. Their plasma concentrations of 17-OHP at screening were 243 and 230 nmol/l. Their CAH diagnosis was confirmed by urine steroid hormone analysis and elevated serum 17-OHP (230 and 113 nmol/l).
False-positive cases
With use of the gestational age-related cut-off limits the overall false-positive rate was 0.03% (1 : 4000) (Table 2). In spite of the gestational age-related limits, 67% of all false-positive cases were born preterm. Their mean gestational age was 28 weeks (range: 24-36). The majority of the false-positive infants were hospitalized in neonatal wards at the time of the alert.
164
False-negative cases
Four missed cases of CAH were identified in the screened population. Case I. This girl was born at term. Her screening sample contained ~40 nmol
17-OHP/l plasma. She was diagnosed at the age of 2.5 years because of growth acceleration and clitoris enlargement (present from birth according to the mother). She had had three episodes of fever and vomiting, requiring intravenous rehydration on one occasion. No abnormal serum electrolyte levels were recorded. The CAH diagnosis was confirmed by the urine steroid hormone profile. She was considered to have simple virilizing CAH. Case ZZ. This girl was born at term. She had clitoris enlargement and labial fusion. Serum Na was 133-137 and K 7.4 mmol/l. The concentration of 17-OHP in her screening sample on day 4 was 118 nmol/l plasma. A second filter paper sample on day 11 gave a value of 394 nmol of 17-OHP/l plasma (323 nmol/l after ether extraction) and a serum sample on the same day of 249 nmol/l serum. The CAH diagnosis was confirmed by the urine steroid hormone pattern and treatment was started on day 19. Case ZZZ. This girl had an elder sister with late-diagnosed mild CAH. This information was unfortunately not conveyed to the screening laboratory. The girl was born at term and was not virilized. Her neonatal screening sample was negative, i.e., the 17-OHP concentration was 152 nmol/l plasma (cut-off limit at that time 200 nmol/l plasma). After the age of 1.5 years she developed clitoris enlargement, pubarche and accelerated skeletal maturation (bone age was + 3.5 SD at 2 years of age). Her basal serum 17-OHP at the age of 2 years was 800 nmol/l and after ACTH stimulation it increased markedly to 1500 nmol/l with a small serum cortisol increase (from 164 to 260 nmol/l). Treatment was begun at the age of 2.6 years. Case IV. This girl was born at term. She had clitoris enlargement and urogenital sinus and CAH was therefore suspected. Serum electrolytes were normal. Her screening sample on day 5 contained 125 nmol 17-OHP/l plasma (cut off limit 150). Therapy was started on day 10 after an analysis performed at the local laboratory had shown a serum 17-OHP concentration of 92 nmol/l (reference range: < 4 nmol/l). Unscreened CAH patient
One Swedish child with CAH was not screened because she was born abroad after 28 weeks of gestation. She was initially considered to be a boy with hypospadia and cryptorchidism. The child developed significant hyponatremia (lowest serum sodium 126 mmol/l). The CAH diagnosis was established at the age of 25 days. The child’s gender was then corrected and hormone supplementation was initiated.
Discussion
The present study comprised more than 0.5 million newborn infants screened for CAH. The goal of the screening was to detect patients with CAH before they were diagnosed on grounds of serious symptoms. More than 90% of all known CAH
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patients were diagnosed before the age of 2 weeks. This is considerably earlier than was the case before screening was started [12]. The screening analysis led to earlier diagnosis in 52% of the CAH patients, whereas in the remaining 48% - mostly girls - the diagnosis had already been made or was highly suspected at the time when the screening result became available. The median age at which the CAH diagnosis in the screened population was established and treatment with cortisone was begun was 12 days, compared with 30 days before screening [12]. By screening it was possible to avoid serious salt-loss crises after the age of 2 weeks. Incorrect gender assignment is a problem in girls with CAH [12]. There was considerable delay in correct gender assignment before screening, but not after. Additional benefits of CAH screening are that virilization, growth acceleration and prernature pubarche are decreased. An imeresting observation was that the earlier mentioned nonvirilized girl with CAH whose mother was treated with dexamethasone 1.5-1.75 mg daily from the sixth week of gestation to the end of pregnancy nevertheless had elevated 17-OHP in her screening sample collected 3 days after birth; she was born at term and had more than 700 nmol of 17-OHP/l of plasma before and 574 nmol/l after extraction of the sa.mple with ether. A similar case was reported earlier [3]. Congenital adrenal hyperplasia is a very heterogeneous condition [2,14]. Multiple mutant 21-hydroxylase alleles have now been identified. It is likely that there is a continuum of deficiencies rather than only two categories of CAH as assumed previously, i.e., classical CAH and the late-onset or cryptic type. It is therefore not surprising that a screening system will have occasional false-negative test results. We encountered four such cases. It is also possible that patients with mild CAH, missed by screening, will be identified in the future. The false-positive group consisted mainly of preterm infants, in spite of the use of gestational age-related cut-off limits. The infants were usually still in neonatal wards at the time of the alert and a second follow-up sample could easily be obtained. We conclude that the benefits of CAH screening are significant and recommend adding CAH screening to the neonatal metabolic screening program in Sweden, which would then include PKU, galactosemia, congenital hypothyroidism and CAH.
Acknowledgement This stu’dy was supported by grants from the Swedish Medical Research Council (4792).
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