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Prenatal diagnosis of fetal cytomegalovirus infection
Prenatal diagnosis of fetal cytomegalovirus infection Monique E. Lamy, MD,. Kipanga N. Mulongo, MD,. Jean-Fram,ois Gadisseux, MD," Gilbert Lyon, MD,b Veronique Gaudy, MD,c and Michel Van Lierde, MDc Brussels, Belgium A prospective study (1988 to 1990) to evaluate the risk of fetal cytomegalovirus transmission was conducted with 1771 pregnant women; the test results of 861 were seronegative (48.6%). At each prenatal visit they were tested for serologic data and cytomegalovirus excretion in urine, saliva, and cervical secretions. If seroconverslon occurred (with or without cytomegalovirus excretion), ultrasonography, amniocentesis, and cordocentesis were performed at 22 weeks' gestation; 7 cases of primary cytomegalovirus infection were investigated. In 5 cases the amniotic fluid cultures were positive; in 3 cases the fetal blood test results were positive for specific immunoglobulin M; and in 2 cases brain ultrasonography results were positive. Infection was confirmed with biopsies of fetal tissue. In two other cases, the cord blood and amniotic fluid test results were negative, and the neonates were free of infection. (AM J OBSTET GVNECOL 1992;166:91-4.)
Key words: Prenatal, cytomegalovirus, amniocentesis, cordocentesis, ultrasonography Cytomegalovirus is considered to be the first cause of congenital infection. 1 Maternal infections are mostly asymptomatic; however, transplacental transmission occurs in about 50% of cases. 2 Furthermore, 90% of infected neonates show no clinical signs of infection at birth, although some will have late manifestations of
congenital cytomegalovirus infection? Prenatal diagnosis is therefore essential to detect those fetuses at risk of having symptoms and late sequelae. In a prospective survey seven cases of primary cytomegalovirus infection were investigated for fetal transmission. In addition to detecting specific immunoglobulin M (IgM) antibodies with cord blood tests and fetal brain echography, we emphasized virus isolation from amniotic fluid culture for antenatal diagnosis.
From the Departments oJVzrolo/sy," NeUlopediatno,b and ObstetrzC5,' Catholzc UnivPrslly oj Louvaln. Suppmted by a grant Jrom the Marguerite Marze Delacrozx Foundation and by Fonds National de Recherche Sczentzjique and Fonds de Recherche SClentifique Medzcale Conventzon Jor Neuropatholo/sy Studzes. RecezvedJor publzcatlOnJanuary 8,1991: revz;ed May 9,1991: accepted July 16, 1991. Reprint request I: Monzque E. Lamy, MD, Department oj Vlrolo/sy, E.5.P., U C.L., 30155 Clos Chapelle aux Champs, 1200 Brussels, Belglum. 611 132433
Material and methods Patients. A prospective study was conducted with 1771 pregnant women; the test results of 861 were seronegative (48.6%). At each prenatal visit they were tested for serologic data and cytomegalovirus excretion in urine, saliva, and cervical secretions. If sero-
Table I. Prenatal diagnosis of cytomegalovirus infection Cordocenteszs
Case No.
Age (yr)
G,P
GestatIOnal age at przmary InJectlOn (wh)
I 2
27 29
G2, PI G2, PI
7-15 8-12
3
26
G3, P2
9-13
4
27
G2, PI
<9
5
25
GI, PO
5-10
1:20
6 7
29 30
G2, PI G3, PI
<9 13-18
<1:40
IgM
I: 160
I
IgG
AmnzocenteslS (amnlOtic flUId CMV C1llture)
Brain ultrasonography result
I: 320
+
10' PFU/ml 10' PFUlml
1:640
10" PFUlml
I: 160
10 5 PFU/ml
+
I: 20,480
10 7 PFU/ml
+
+
1:1280 1:320
G, Gravida; p, para; CMV, cytomegalovirus; PFU, plaque-forming units.
91
92
Lamy et al.
January 1992 Am J Obslel Gynecol
Table II. Outcome of pregnancy and findings at termination or delivery Fetlls Bram microscopic
Outcome of pl'egnancy
PositIVe CMV Isolation
PositIVe CMV mcillSions or antigen
2
I
TOP TOP
Amniotic fluid, liver, spleen Amniotic fluid, liver
1,2 1,2
3
TOP
4
TOP
Brain. lung, liver, pancreas. kidneys
1-5
5
TOP
Brain, thyroid, lungs, liver. pancreas, kidn ey;, adrenal glands
1-4.6
6
Born Born
Amniotic fluid, placenta, heart, liver, spleen, eye Amniotic fluid , placenta, liver, spleen, eye Amniotic fluid, placenta, heart, lung, kidney, liver, spleen, eye
Brain, thyroid, liver, pancreas, kidneys Brain, eye, thyroid, lung, liver, spleen, adrenal glands, pancreas, kidneys Brain
Case No.
7
le~lOns*
Neonates (unne and salIVa at bUlit)
1.2
CMV, Cytomegalovirus; TOP, termination of pregnancy. *LeslOn 1, Inflammatory infiltrate; 2, microglial nodules; 3, focal necrosis; 4. calcifications; 5, cortical abnormali ties, 6, ependymitis.
conversion occurred (with or without cytomegalovirus excretion), ultrasonography, amniocentesis, and cordocentesis were performed at 22 weeks' gestation with informed consent from these women to detect transplacental cytomegalovirus transmission. Serologic methods. Sera or fetal blood was titrated for IgM and immunoglobulin G (IgG) by enzymelinked immunosorbent assay (Behring, Marburg, Germany) and for complement-fixing antibodies (antigen virion). Cytomegalovirus isolation. Samples of urine , saliva, cervical secretions, and amniotic fluid were filtered through a 0.45 ILm Minisart; 0.2 ml of the sample was inoculated in duplicate on human embryonic lung cells (MRC5 cell line, Biomerieux, Lyon, France), centrifuged at 3000 rpm for 45 minutes in macroplates (Nuncion, Roskilde, Denmark), and incubated at 37 C. After 48 hours, replication of cytomegalovirus was detected by immunoperoxidase staining with a monoclonal antibody to cytomegalovirus immediate early antigen (Biosoft, Paris, France). The same samples were cultured on tubes to detect characteristic cytopathic effect. 0
Results Seroconversion occurred in 20 of 861 seronegative pregnant women (2.3%). Seven of 20 agreed to cordocentesis and amniocentesis. The mean age of the seven patients was 27.5 years. They were all asymptomatic, except for the one who presented with clinical and hematologic signs of mononucleosis . In five cases (Table 1, cases 1 to 5), amniotic fluid culture yielded a high titer of virus (up to 10' plaque-forming units per milliliter). The fetal blood test results were positive in three
cases (I, 2, 5) for specific IgM antibodies and negative in the two other cases (3, 4). Brain microcalcifications were visualized in two cases (4. 5) by ultrasonography. Our ethical committee approved the termination of pregnancies requested by these five patients. Therapeutic abortions were achieved by intraamniotic injection of prostaglandin E2 and intravenous injection of oxytocin. Afterward the placenta was systematically manually separated and removed. After the pregnancies were terminated, evidence of infection was confirmed either by positive culture or by demonstrating cytomegalovirus antigen or inclusions on histologic sections of various organs (brain, eye, thyroid , heart, lungs, liver, spleen, pancreas, adrenal glands, kidne ys) (Table II). In all five cases microscopic cerebral lesions consisted of inflammatory changes with perivascular infiltrates, focal areas of ependymal disruption, and glial nodules (Fig. 1, B). Calcified necrotic areas (Fig. 1, A) were observed in the periventricular region in cases 4 and 5. Microglial and neuronal cells containing cytomegalovirus inclusions were identified with immunohistochemical stain (Fig. 1). These cells were widely distributed, more abundantly in the brain stem, cerebellum , and entorhinal regions. In addition , discrete cortical abnormalities were observed (case 4) with microgyric-like cortical foldings. In the two remaining pregnancies (cases 6 and 7) no virus was isolated from amniotic fluid culture and no specific IgM antibodies could be found in cord blood. After deli very at term, the newborns were free of infection: no specific IgM antibodies were detected in their serum, and they did not shed virus fro m urine or saliva within the first 2 weeks of life.
Prenatal diagnosis of cytomegalovirus
Volume 166 Number I, Part I
A
,
.
..
.
Fig. 1. Brain lesions in cytomegalovirus-infected fetuses. A, Perivascular calcified necrotic area and focal ependymal disruptions (arrows) by inflammatory cells; coronal section through the entorhinal region. (Hematoxylin-eosin stain; x 12.) B, Inflammatory nodule with intracytoplasmic cytomegalic inclusion (arrows), subcortical region. (Hematoxylin-eosin stain; x 150.) C, Nuclear cytomegalic inclusions revealed by using an immunoperoxidase staining with anticytomegalovirus monoclonal antibody. Neocortical region. (Hematoxylin-eosin counterstain; x 180.)
93
94
Lamy et al.
Comment Detection of specific IgM in fetal blood was considered to be the best marker for prenatal diagnosis of cytomegalovirus infection. 3. 7 These antibodies are detected only after 20 weeks of pregnancy." Furthermore, an immature immune system or insufficient test sensitivity could lead to a false-negative result of IgM. 9 We found amniotic fluid culture to be a particularly sensitive indicator of cytomegalovirus transmission. It succeeded in predicting infection in five fetuses, confirmed by viral isolation from various organs and histologic lesions, which included the central nervous system; whether these lesions are responsible for neurologic sequelae requires further studies. Amniotic fluid culture also proved its specificity by showing negative results in the two cases free of infection at birth. We advocate that amniotic fluid culture be used in any case of suspected congenital infection. Two recently published reports dealing with amniocentesis corroborate our observations!o.!!; however, more amniocenteses performed before 22 weeks are needed to assess how early a marker amniotic fluid culture can be. We thank M. Pirenne, M. Lebyn, M. Peers, L. Croonen, and B. Van De Put for their skillful technical assistance.
REFERENCES 1. Remington JS, Klein JO. Infectious diseases of the fetus
and newborn infant. Philadelphia: WB Saunders, 1990: 241-81.
January 1992 Am J Obstet Gynecol
2. Fields NB, Alford CHA, Britt WJ. Cytomegalovirus. In: Virology. New York: Raven Press, 1990:1981-2010. 3. Stagno S, Pass RF, Reynolds DW, Moore M, Nahmias AJ, Alford CA. Comparative study of diagnostic procedures for congenital cytomegalovirus infection. Pediatrics 1980;65:251-7. 4. Stagno S, Pass RF, Thomas JP, Navia JM, Dworsky MD. Defects of tooth structure in congenital cytomegalovirus infection. Pediatrics 1982;69:646-8. 5. Stagno S, Reynolds DW, Amos CS, et at. Auditory and visual defects resulting from symptomatic and subclinical congenital cytomegaloviral and toxoplasma infections. Pediatrics 1977;59:669-78. 6. Stagno S, Reynolds DW, Pass RF, Alford CA. Breast milk and the risk of cytomegalovirus infection. N Engl J Med 1980;302: 1073-6. 7. Stagno S, Reynolds DW, Tsiantos A, Fuccillo DA, Long W, Alford CA Jr. Comparative, serial virologic and serologic studies of symptomatic and subclinical congenital and natally acquired cytomegalovirus infection. J Infect Dis 1975;132:568-77. 8. Voisin GA, Edelman P, Genetet N, Bach JF, Sureau C. Immunologie de la reproduction. Paris: Flammarion, 1990:439-45. 9. Ahlfors K, Forsgren M, Griffiths p, Nielsen CM. Comparison of four serological tests for the detection of specific immunoglobulin M in cord sera of infants congenitally infected with cytomegalovirus. Scand J Infect Dis 1987;19:303. 10. Meisel R. Fetal cytomegalovirus infection: a case report. AMJ OBSTET GYNECOL 1990;162:663-4. 11. Weiner CP, Grose C. Prenatal diagnosis of congenital cytomegalovirus infection: 2 decades later. AM J OBSTET GYNECOL 1990;163:447-50.
Key words: Prenatal, cytomegalovirus, amniocentesis, cordocentesis, ultrasonography Cytomegalovirus is considered to be the first cause of congenital infection. 1 Maternal infections are mostly asymptomatic; however, transplacental transmission occurs in about 50% of cases. 2 Furthermore, 90% of infected neonates show no clinical signs of infection at birth, although some will have late manifestations of
congenital cytomegalovirus infection? Prenatal diagnosis is therefore essential to detect those fetuses at risk of having symptoms and late sequelae. In a prospective survey seven cases of primary cytomegalovirus infection were investigated for fetal transmission. In addition to detecting specific immunoglobulin M (IgM) antibodies with cord blood tests and fetal brain echography, we emphasized virus isolation from amniotic fluid culture for antenatal diagnosis.
From the Departments oJVzrolo/sy," NeUlopediatno,b and ObstetrzC5,' Catholzc UnivPrslly oj Louvaln. Suppmted by a grant Jrom the Marguerite Marze Delacrozx Foundation and by Fonds National de Recherche Sczentzjique and Fonds de Recherche SClentifique Medzcale Conventzon Jor Neuropatholo/sy Studzes. RecezvedJor publzcatlOnJanuary 8,1991: revz;ed May 9,1991: accepted July 16, 1991. Reprint request I: Monzque E. Lamy, MD, Department oj Vlrolo/sy, E.5.P., U C.L., 30155 Clos Chapelle aux Champs, 1200 Brussels, Belglum. 611 132433
Material and methods Patients. A prospective study was conducted with 1771 pregnant women; the test results of 861 were seronegative (48.6%). At each prenatal visit they were tested for serologic data and cytomegalovirus excretion in urine, saliva, and cervical secretions. If sero-
Table I. Prenatal diagnosis of cytomegalovirus infection Cordocenteszs
Case No.
Age (yr)
G,P
GestatIOnal age at przmary InJectlOn (wh)
I 2
27 29
G2, PI G2, PI
7-15 8-12
3
26
G3, P2
9-13
4
27
G2, PI
<9
5
25
GI, PO
5-10
1:20
6 7
29 30
G2, PI G3, PI
<9 13-18
<1:40
IgM
I: 160
I
IgG
AmnzocenteslS (amnlOtic flUId CMV C1llture)
Brain ultrasonography result
I: 320
+
10' PFU/ml 10' PFUlml
1:640
10" PFUlml
I: 160
10 5 PFU/ml
+
I: 20,480
10 7 PFU/ml
+
+
1:1280 1:320
G, Gravida; p, para; CMV, cytomegalovirus; PFU, plaque-forming units.
91
92
Lamy et al.
January 1992 Am J Obslel Gynecol
Table II. Outcome of pregnancy and findings at termination or delivery Fetlls Bram microscopic
Outcome of pl'egnancy
PositIVe CMV Isolation
PositIVe CMV mcillSions or antigen
2
I
TOP TOP
Amniotic fluid, liver, spleen Amniotic fluid, liver
1,2 1,2
3
TOP
4
TOP
Brain. lung, liver, pancreas. kidneys
1-5
5
TOP
Brain, thyroid, lungs, liver. pancreas, kidn ey;, adrenal glands
1-4.6
6
Born Born
Amniotic fluid, placenta, heart, liver, spleen, eye Amniotic fluid , placenta, liver, spleen, eye Amniotic fluid, placenta, heart, lung, kidney, liver, spleen, eye
Brain, thyroid, liver, pancreas, kidneys Brain, eye, thyroid, lung, liver, spleen, adrenal glands, pancreas, kidneys Brain
Case No.
7
le~lOns*
Neonates (unne and salIVa at bUlit)
1.2
CMV, Cytomegalovirus; TOP, termination of pregnancy. *LeslOn 1, Inflammatory infiltrate; 2, microglial nodules; 3, focal necrosis; 4. calcifications; 5, cortical abnormali ties, 6, ependymitis.
conversion occurred (with or without cytomegalovirus excretion), ultrasonography, amniocentesis, and cordocentesis were performed at 22 weeks' gestation with informed consent from these women to detect transplacental cytomegalovirus transmission. Serologic methods. Sera or fetal blood was titrated for IgM and immunoglobulin G (IgG) by enzymelinked immunosorbent assay (Behring, Marburg, Germany) and for complement-fixing antibodies (antigen virion). Cytomegalovirus isolation. Samples of urine , saliva, cervical secretions, and amniotic fluid were filtered through a 0.45 ILm Minisart; 0.2 ml of the sample was inoculated in duplicate on human embryonic lung cells (MRC5 cell line, Biomerieux, Lyon, France), centrifuged at 3000 rpm for 45 minutes in macroplates (Nuncion, Roskilde, Denmark), and incubated at 37 C. After 48 hours, replication of cytomegalovirus was detected by immunoperoxidase staining with a monoclonal antibody to cytomegalovirus immediate early antigen (Biosoft, Paris, France). The same samples were cultured on tubes to detect characteristic cytopathic effect. 0
Results Seroconversion occurred in 20 of 861 seronegative pregnant women (2.3%). Seven of 20 agreed to cordocentesis and amniocentesis. The mean age of the seven patients was 27.5 years. They were all asymptomatic, except for the one who presented with clinical and hematologic signs of mononucleosis . In five cases (Table 1, cases 1 to 5), amniotic fluid culture yielded a high titer of virus (up to 10' plaque-forming units per milliliter). The fetal blood test results were positive in three
cases (I, 2, 5) for specific IgM antibodies and negative in the two other cases (3, 4). Brain microcalcifications were visualized in two cases (4. 5) by ultrasonography. Our ethical committee approved the termination of pregnancies requested by these five patients. Therapeutic abortions were achieved by intraamniotic injection of prostaglandin E2 and intravenous injection of oxytocin. Afterward the placenta was systematically manually separated and removed. After the pregnancies were terminated, evidence of infection was confirmed either by positive culture or by demonstrating cytomegalovirus antigen or inclusions on histologic sections of various organs (brain, eye, thyroid , heart, lungs, liver, spleen, pancreas, adrenal glands, kidne ys) (Table II). In all five cases microscopic cerebral lesions consisted of inflammatory changes with perivascular infiltrates, focal areas of ependymal disruption, and glial nodules (Fig. 1, B). Calcified necrotic areas (Fig. 1, A) were observed in the periventricular region in cases 4 and 5. Microglial and neuronal cells containing cytomegalovirus inclusions were identified with immunohistochemical stain (Fig. 1). These cells were widely distributed, more abundantly in the brain stem, cerebellum , and entorhinal regions. In addition , discrete cortical abnormalities were observed (case 4) with microgyric-like cortical foldings. In the two remaining pregnancies (cases 6 and 7) no virus was isolated from amniotic fluid culture and no specific IgM antibodies could be found in cord blood. After deli very at term, the newborns were free of infection: no specific IgM antibodies were detected in their serum, and they did not shed virus fro m urine or saliva within the first 2 weeks of life.
Prenatal diagnosis of cytomegalovirus
Volume 166 Number I, Part I
A
,
.
..
.
Fig. 1. Brain lesions in cytomegalovirus-infected fetuses. A, Perivascular calcified necrotic area and focal ependymal disruptions (arrows) by inflammatory cells; coronal section through the entorhinal region. (Hematoxylin-eosin stain; x 12.) B, Inflammatory nodule with intracytoplasmic cytomegalic inclusion (arrows), subcortical region. (Hematoxylin-eosin stain; x 150.) C, Nuclear cytomegalic inclusions revealed by using an immunoperoxidase staining with anticytomegalovirus monoclonal antibody. Neocortical region. (Hematoxylin-eosin counterstain; x 180.)
93
94
Lamy et al.
Comment Detection of specific IgM in fetal blood was considered to be the best marker for prenatal diagnosis of cytomegalovirus infection. 3. 7 These antibodies are detected only after 20 weeks of pregnancy." Furthermore, an immature immune system or insufficient test sensitivity could lead to a false-negative result of IgM. 9 We found amniotic fluid culture to be a particularly sensitive indicator of cytomegalovirus transmission. It succeeded in predicting infection in five fetuses, confirmed by viral isolation from various organs and histologic lesions, which included the central nervous system; whether these lesions are responsible for neurologic sequelae requires further studies. Amniotic fluid culture also proved its specificity by showing negative results in the two cases free of infection at birth. We advocate that amniotic fluid culture be used in any case of suspected congenital infection. Two recently published reports dealing with amniocentesis corroborate our observations!o.!!; however, more amniocenteses performed before 22 weeks are needed to assess how early a marker amniotic fluid culture can be. We thank M. Pirenne, M. Lebyn, M. Peers, L. Croonen, and B. Van De Put for their skillful technical assistance.
REFERENCES 1. Remington JS, Klein JO. Infectious diseases of the fetus
and newborn infant. Philadelphia: WB Saunders, 1990: 241-81.
January 1992 Am J Obstet Gynecol
2. Fields NB, Alford CHA, Britt WJ. Cytomegalovirus. In: Virology. New York: Raven Press, 1990:1981-2010. 3. Stagno S, Pass RF, Reynolds DW, Moore M, Nahmias AJ, Alford CA. Comparative study of diagnostic procedures for congenital cytomegalovirus infection. Pediatrics 1980;65:251-7. 4. Stagno S, Pass RF, Thomas JP, Navia JM, Dworsky MD. Defects of tooth structure in congenital cytomegalovirus infection. Pediatrics 1982;69:646-8. 5. Stagno S, Reynolds DW, Amos CS, et at. Auditory and visual defects resulting from symptomatic and subclinical congenital cytomegaloviral and toxoplasma infections. Pediatrics 1977;59:669-78. 6. Stagno S, Reynolds DW, Pass RF, Alford CA. Breast milk and the risk of cytomegalovirus infection. N Engl J Med 1980;302: 1073-6. 7. Stagno S, Reynolds DW, Tsiantos A, Fuccillo DA, Long W, Alford CA Jr. Comparative, serial virologic and serologic studies of symptomatic and subclinical congenital and natally acquired cytomegalovirus infection. J Infect Dis 1975;132:568-77. 8. Voisin GA, Edelman P, Genetet N, Bach JF, Sureau C. Immunologie de la reproduction. Paris: Flammarion, 1990:439-45. 9. Ahlfors K, Forsgren M, Griffiths p, Nielsen CM. Comparison of four serological tests for the detection of specific immunoglobulin M in cord sera of infants congenitally infected with cytomegalovirus. Scand J Infect Dis 1987;19:303. 10. Meisel R. Fetal cytomegalovirus infection: a case report. AMJ OBSTET GYNECOL 1990;162:663-4. 11. Weiner CP, Grose C. Prenatal diagnosis of congenital cytomegalovirus infection: 2 decades later. AM J OBSTET GYNECOL 1990;163:447-50.