Vohm~e 93 Number 2
25.
26.
27.
28.
29.
30.
estal~lishment of the neonatal bacterial flora. I. The role of host factors, Arch Environ Health 21:514, 1970. McAllister TA, G-ivan J, Black A, Turner M J, Kerr bib,l, and tlutchinson JH: The natural history of bacterial colonizatioh of the newborn in a maternity hospital (Part 1), Scot Med J 19:119, 1974. Long SS, and Swenson RM: Development of an~ierobic fecal flora in healthy newborn infants, J Prblx'rR 91:298, 1977. Gothefors L, Carlsson B, Ahlstedt S, Harrison LA, and Winberg J: Influence of maternal gut flora and colostral a n d cord seruni an!ibodies on presence of F_scherichia coil in feces of the newborn infant, Acta Pediatr Scand 65:225, 1976. Tessier F, and Daguet GL: Influence des anfibi0tiques sur les efit,~robactrries de la flore intestinale au cours de la p~riode n~onatale, Therapeutique 48:47, 1972. McFarlan AM, Crone PB, and Tee GH: Variations in bacteriology of throat and rectum of inf,'ints in two maternity.units, Br Med .I 2:1140, i949. Johanson WG, Pierce AK, Sanford JD, and Thomas GD: Nosbcomial respiratory infections With gram-negative bacilli, Ann Intern Med 77:701, 1972.
Bacterial colonization in N I C U
293
31. Sprunt K, Leidy G, and Redman W: Implantation to prevent infection in neonatal ICU, Pediatr Res 9:345, 1975. 32. Selden C, Lee S, Wang WLL, Bennett JV, and EickhoffTC: Nosocomial Klebsiella infections: intestinal colonization as a reservoir, Ann Intern Med 74:657, 1971. 33. Shinefield HR., Ribble JC, and Boris M: Bacterial interference between strains of Staphylococcus aureus, 1960 to i9"/0, Am J Dis Child 121:148, 1971. 34. Sprunt K, Leidy G, and Redman W: Pharyngeal implantation of alpha hemolytic streptococci (a-strep) in neonates in an ICU, Pediatr Res 11:506, 1977. 35. Sears HJ, Jones H, Saloum R, Brownlee I, and Lamoreaux LF: Persistence of individual strains of Escherichia coil in na:~n~ind dog under varying conditions, J Bacteriol 24:235, 1964. 36. Formal SB, Dammin G, Spinz ti, Kundel D, Schneider H, Hoi'owitz PE, and Forbes M: Experimental shigella infections. V. Studies in germ-free guinea pigs, J Bacteriol 82:i84, 1961. 37. Houck PW, Nelson JD, and Kay JL: Fatal septicemia due to Staphylococcus aureus 502A; report of a case and review of the infectious complications of bacterial interference programs, Am J Dis Child 123:45, 1972.
Brief clinical and laboratory observation Prenatal diagnosis in twin pregnancy Yoshi)uki Suzuki, M.D.,* Norimasa Nakamura, M.D., Toshlharu ,limbo, M.D, Sadao ltoriguchi, M.D., and Toshl Fujll, M.D., Tokyo, Japan
ASSAYS OF ENZYME ACTIVITIES in cultured amniotic fluid cells have been used for the prenatal diagnosis of i n b o r n errors of metabolism. Hox;eever, a problem occurs when the fetuses are tWirls. In a case of high-risk preg-
From the Departments of Pediatrics and Obstetrics, Faculty of 31edicine, Uni~'ersit)"of Tokyo, and Departments of Obstetrics and Pediatrics, Tokyo Metropolitan Tsukiji Maternity Hospital Supported by the grantsfronz the Ministry of Education, and the Mhzistry of Health and Welfare of Japan. *Reprintaddress:Departmentof Pediatrics, Facultyof Medicine. Universityof Tokyo, Hongo, Bunk)'o-ku. Tokyo 113. Japan.
0022-3476/78/0293-0293500.20/0 9 1978 The C. V. Mosby Co.
nancy for globoid cell leukodystrophy, dizygotic twins were found after the therapeutic abortion. One of them was affected with this disease. Abbreviations used GLD: globoid cell leukodystrophy 4MU: 4-methylumbelliferyl CMH: galactocerebroside MATERIALS
AND METIIOI)S
The parents of a patient who had died of GLD were referred for the purpose of prenatal diagnosis o f the fetus during the second pregnancy. The diagnosis in the first child was established by demonstration ofgalactocerebrosidase deficiency in plasma and leukocytes. Amniocen-
294
Brief clinical and laboratoQ' observation
The Journal of Pediatrics August 1978
Table I. fl-Galactosidases in fetal tissues
Brain CMII Fetuses at risk 1 (23 wk) 2 Control fetuses 1 (21 wk) 2 (18 wk)
I 4MU
I
Liver CMH [
4MU
0.48 0.03
52 86
0.26 0.02
241 375
1.62 0.72
96 45
0.73 0.44
451 280
= Galactocerebrosidase;4MU = 4-meth)'lumbelliferylfl-galactosidase; eazymeunit 9 nmol/mg protein/hour. CMIt
tesis was carried out at 18 weeks of gestation. The amniotic fluid ~:ells were cultured in F-10 culture medium for five weeks, were harvested by scraping off, and were washed three times with isotonic saline. They were ultrasonically disrupted in water and used for enzyme assay. fl-Galactosidase activities were assayed as reported previously, ~ Using 4-methylumbelliferyl fl-galactopyranoside (Nakarai Chemicals, Ltd., Kyoto, Japan) and tritiumlabeled galactocerebroside as substrates. The protein content was determined by the method of Lowry et al.: The autopsy tissues of the aborted twin fetuses were stored frozen at - 2 0 ~ until chemical analysis. The presence of twins was not anticipated preoperatively, although sonography was used for the identification of placenta and fetal parts. They were dizygotic according to the morphology of the placenta. Brain and liver tissues were examined enzymatically. The tissues were homogenized in water at a concentration of 100 mg/ml, disrupted by Sonication, centrifuged at 1,000 • g for 10 minutes, and the supernatant was used for enzyme assay. The control tissues were obtained from fetuses aborted for maternal indications. RESULTS A specific deficiency ofgalactocerebrosidase was found in the amniotic fluid cells (fetus at risk 0.01 nmol/mg protein/hour, and a control 0.36 n m o l / m g protein/hour). Activities of other lysosomal hydrolases, including flgalactosidases, toward synthetic substrates were as high as those in the control cells. As shown in Table I, the enzyme assays revealed low galactocerebrosidase activity in brain and liver of the first fetus, and a profound deficiency of this enzyme in the tissues of the second fetus, confirming the results Of the prenatal diagnosis in the latter. It is highly likely that the first fetus was a heterozygous carrier of this disease. DISCUSSION Three cases of fetal GLD have been described. :'-'' This report confirms the reliability of the prenatal diagnosis of
GLD by assay of galactocerebrosidase activity in cultured amniotic fluid cells, although it was complicated by twin pregnancy. The diagnosis of fetal GLD was established by the enzyme assay on the tissues from one of the fetuses. The other fetus was probably a GLD carrier. If the amniotic fluid ceils had originated from the latter, the fetus with. t3LD might not have been identified. To our knowledge there are no previously reported instances of prenatal diagnosis o f inborn metabolic diseases in one of dizygotic twins. A twin case of Down syndrome has been rePorted recently. ~ Twin pregnancy seems not to have been anticipated in that case either. The use of sonography w a s n o t mentioned. Itowever, if twins are found before amniocentesis in high-risk pregnancy, amniotic fluid should be drawn from each amniotic sac individually, as reported by Bang et al. s The ethical aspect o f prenatal diagnosis in twin pregnancy desexes discussion. In this case the parents wished the termination of pregnancy as the enzyme assay o f the cultured amniotic fluid cells indicated the fetus to be affected by GLD. If the presence of twins had been found by preoperative procedures, a potential conflict would have occurred \vith regard to the treatment o f the fetuses. It is generally agreed, however, that the final decision should be made by the parents. The tritium-labeled galactocerebroside was kindly donated by Dr. S. Handa, Department of Biochemistry, Faculty of Medicine, University of Tokyo. REFERENCES I. Suzuki Y, and Suzuki K: Krabbe's globoid cell leukodystrophy: Deficiency of galactocerebrosidase in serum, leukocytes and fibroblasts, Science 171:73, 1971. 2. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent, J Biol Chem 193:265, 1951. 3. Suzuki K, Schneider EL, and Epstein CJ: In utero diagnosis of globoid cell leukodystrophy (Krabbe's disease), Biochem Biophys Res Commun 45:1363, 1971. 4. Ellis WG, Schneider EL, McCulloch JR, Suzuki K; and Epstein CJ: Fetal globoid cell leukodystrophy (Krabbe disease), Arch Neurol 29:253, 1973. 5. Harzcr K, Benz HU, Kn6rr-GSrtner tt, Jonatha WD, and Kn6rr K: Pr~inatale Diagnose der GloboidzelI-Leukodystrophie (Morbus Krabbe), Dtsch Med Wochenschr 101:821, 1976. 6. Harzer K: Prenatal diagnosis ofgl0boid cell leukodystrophy (Krabbe's disease). Third documented case, llum Genet 35:193, 1977. 7. Leschot NJ, Verjaai M, Gravenhorst JB, and van de Kamp JJP: An unusual case of prenatal di~ignosis in twin pregnancy, Clin Genet 11:441, 1977. 8. Bang J, Nielsen H, and Philip J: Prenatal karyotyping of twins by ultrasonically guided amniocentesis, Am J Obstet Gynecol 123:695, 1975.
25.
26.
27.
28.
29.
30.
estal~lishment of the neonatal bacterial flora. I. The role of host factors, Arch Environ Health 21:514, 1970. McAllister TA, G-ivan J, Black A, Turner M J, Kerr bib,l, and tlutchinson JH: The natural history of bacterial colonizatioh of the newborn in a maternity hospital (Part 1), Scot Med J 19:119, 1974. Long SS, and Swenson RM: Development of an~ierobic fecal flora in healthy newborn infants, J Prblx'rR 91:298, 1977. Gothefors L, Carlsson B, Ahlstedt S, Harrison LA, and Winberg J: Influence of maternal gut flora and colostral a n d cord seruni an!ibodies on presence of F_scherichia coil in feces of the newborn infant, Acta Pediatr Scand 65:225, 1976. Tessier F, and Daguet GL: Influence des anfibi0tiques sur les efit,~robactrries de la flore intestinale au cours de la p~riode n~onatale, Therapeutique 48:47, 1972. McFarlan AM, Crone PB, and Tee GH: Variations in bacteriology of throat and rectum of inf,'ints in two maternity.units, Br Med .I 2:1140, i949. Johanson WG, Pierce AK, Sanford JD, and Thomas GD: Nosbcomial respiratory infections With gram-negative bacilli, Ann Intern Med 77:701, 1972.
Bacterial colonization in N I C U
293
31. Sprunt K, Leidy G, and Redman W: Implantation to prevent infection in neonatal ICU, Pediatr Res 9:345, 1975. 32. Selden C, Lee S, Wang WLL, Bennett JV, and EickhoffTC: Nosocomial Klebsiella infections: intestinal colonization as a reservoir, Ann Intern Med 74:657, 1971. 33. Shinefield HR., Ribble JC, and Boris M: Bacterial interference between strains of Staphylococcus aureus, 1960 to i9"/0, Am J Dis Child 121:148, 1971. 34. Sprunt K, Leidy G, and Redman W: Pharyngeal implantation of alpha hemolytic streptococci (a-strep) in neonates in an ICU, Pediatr Res 11:506, 1977. 35. Sears HJ, Jones H, Saloum R, Brownlee I, and Lamoreaux LF: Persistence of individual strains of Escherichia coil in na:~n~ind dog under varying conditions, J Bacteriol 24:235, 1964. 36. Formal SB, Dammin G, Spinz ti, Kundel D, Schneider H, Hoi'owitz PE, and Forbes M: Experimental shigella infections. V. Studies in germ-free guinea pigs, J Bacteriol 82:i84, 1961. 37. Houck PW, Nelson JD, and Kay JL: Fatal septicemia due to Staphylococcus aureus 502A; report of a case and review of the infectious complications of bacterial interference programs, Am J Dis Child 123:45, 1972.
Brief clinical and laboratory observation Prenatal diagnosis in twin pregnancy Yoshi)uki Suzuki, M.D.,* Norimasa Nakamura, M.D., Toshlharu ,limbo, M.D, Sadao ltoriguchi, M.D., and Toshl Fujll, M.D., Tokyo, Japan
ASSAYS OF ENZYME ACTIVITIES in cultured amniotic fluid cells have been used for the prenatal diagnosis of i n b o r n errors of metabolism. Hox;eever, a problem occurs when the fetuses are tWirls. In a case of high-risk preg-
From the Departments of Pediatrics and Obstetrics, Faculty of 31edicine, Uni~'ersit)"of Tokyo, and Departments of Obstetrics and Pediatrics, Tokyo Metropolitan Tsukiji Maternity Hospital Supported by the grantsfronz the Ministry of Education, and the Mhzistry of Health and Welfare of Japan. *Reprintaddress:Departmentof Pediatrics, Facultyof Medicine. Universityof Tokyo, Hongo, Bunk)'o-ku. Tokyo 113. Japan.
0022-3476/78/0293-0293500.20/0 9 1978 The C. V. Mosby Co.
nancy for globoid cell leukodystrophy, dizygotic twins were found after the therapeutic abortion. One of them was affected with this disease. Abbreviations used GLD: globoid cell leukodystrophy 4MU: 4-methylumbelliferyl CMH: galactocerebroside MATERIALS
AND METIIOI)S
The parents of a patient who had died of GLD were referred for the purpose of prenatal diagnosis o f the fetus during the second pregnancy. The diagnosis in the first child was established by demonstration ofgalactocerebrosidase deficiency in plasma and leukocytes. Amniocen-
294
Brief clinical and laboratoQ' observation
The Journal of Pediatrics August 1978
Table I. fl-Galactosidases in fetal tissues
Brain CMII Fetuses at risk 1 (23 wk) 2 Control fetuses 1 (21 wk) 2 (18 wk)
I 4MU
I
Liver CMH [
4MU
0.48 0.03
52 86
0.26 0.02
241 375
1.62 0.72
96 45
0.73 0.44
451 280
= Galactocerebrosidase;4MU = 4-meth)'lumbelliferylfl-galactosidase; eazymeunit 9 nmol/mg protein/hour. CMIt
tesis was carried out at 18 weeks of gestation. The amniotic fluid ~:ells were cultured in F-10 culture medium for five weeks, were harvested by scraping off, and were washed three times with isotonic saline. They were ultrasonically disrupted in water and used for enzyme assay. fl-Galactosidase activities were assayed as reported previously, ~ Using 4-methylumbelliferyl fl-galactopyranoside (Nakarai Chemicals, Ltd., Kyoto, Japan) and tritiumlabeled galactocerebroside as substrates. The protein content was determined by the method of Lowry et al.: The autopsy tissues of the aborted twin fetuses were stored frozen at - 2 0 ~ until chemical analysis. The presence of twins was not anticipated preoperatively, although sonography was used for the identification of placenta and fetal parts. They were dizygotic according to the morphology of the placenta. Brain and liver tissues were examined enzymatically. The tissues were homogenized in water at a concentration of 100 mg/ml, disrupted by Sonication, centrifuged at 1,000 • g for 10 minutes, and the supernatant was used for enzyme assay. The control tissues were obtained from fetuses aborted for maternal indications. RESULTS A specific deficiency ofgalactocerebrosidase was found in the amniotic fluid cells (fetus at risk 0.01 nmol/mg protein/hour, and a control 0.36 n m o l / m g protein/hour). Activities of other lysosomal hydrolases, including flgalactosidases, toward synthetic substrates were as high as those in the control cells. As shown in Table I, the enzyme assays revealed low galactocerebrosidase activity in brain and liver of the first fetus, and a profound deficiency of this enzyme in the tissues of the second fetus, confirming the results Of the prenatal diagnosis in the latter. It is highly likely that the first fetus was a heterozygous carrier of this disease. DISCUSSION Three cases of fetal GLD have been described. :'-'' This report confirms the reliability of the prenatal diagnosis of
GLD by assay of galactocerebrosidase activity in cultured amniotic fluid cells, although it was complicated by twin pregnancy. The diagnosis of fetal GLD was established by the enzyme assay on the tissues from one of the fetuses. The other fetus was probably a GLD carrier. If the amniotic fluid ceils had originated from the latter, the fetus with. t3LD might not have been identified. To our knowledge there are no previously reported instances of prenatal diagnosis o f inborn metabolic diseases in one of dizygotic twins. A twin case of Down syndrome has been rePorted recently. ~ Twin pregnancy seems not to have been anticipated in that case either. The use of sonography w a s n o t mentioned. Itowever, if twins are found before amniocentesis in high-risk pregnancy, amniotic fluid should be drawn from each amniotic sac individually, as reported by Bang et al. s The ethical aspect o f prenatal diagnosis in twin pregnancy desexes discussion. In this case the parents wished the termination of pregnancy as the enzyme assay o f the cultured amniotic fluid cells indicated the fetus to be affected by GLD. If the presence of twins had been found by preoperative procedures, a potential conflict would have occurred \vith regard to the treatment o f the fetuses. It is generally agreed, however, that the final decision should be made by the parents. The tritium-labeled galactocerebroside was kindly donated by Dr. S. Handa, Department of Biochemistry, Faculty of Medicine, University of Tokyo. REFERENCES I. Suzuki Y, and Suzuki K: Krabbe's globoid cell leukodystrophy: Deficiency of galactocerebrosidase in serum, leukocytes and fibroblasts, Science 171:73, 1971. 2. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent, J Biol Chem 193:265, 1951. 3. Suzuki K, Schneider EL, and Epstein CJ: In utero diagnosis of globoid cell leukodystrophy (Krabbe's disease), Biochem Biophys Res Commun 45:1363, 1971. 4. Ellis WG, Schneider EL, McCulloch JR, Suzuki K; and Epstein CJ: Fetal globoid cell leukodystrophy (Krabbe disease), Arch Neurol 29:253, 1973. 5. Harzcr K, Benz HU, Kn6rr-GSrtner tt, Jonatha WD, and Kn6rr K: Pr~inatale Diagnose der GloboidzelI-Leukodystrophie (Morbus Krabbe), Dtsch Med Wochenschr 101:821, 1976. 6. Harzer K: Prenatal diagnosis ofgl0boid cell leukodystrophy (Krabbe's disease). Third documented case, llum Genet 35:193, 1977. 7. Leschot NJ, Verjaai M, Gravenhorst JB, and van de Kamp JJP: An unusual case of prenatal di~ignosis in twin pregnancy, Clin Genet 11:441, 1977. 8. Bang J, Nielsen H, and Philip J: Prenatal karyotyping of twins by ultrasonically guided amniocentesis, Am J Obstet Gynecol 123:695, 1975.
