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Enterovirus in pregnant women and the perfused placenta
Enterovirus in pregnant women and the perfused placenta Marvin S. Amstey, MD, Richard K. Miller, PhD, Marilyn A. Menegus, PhD, and P. Anthony di Sant 'Agnese, MD Rochester, New York A survey of all nonpolio enterovirus infections in adults for a 10-year period revealed that most such infections will occur in the child-bearing age group during the third quarter of the year. Coxsackieviruses occurred more often than echovirus infec~ion. Fourteen pregnant women, including four with meningitis, who had an enterovirus infection from 16 to 37 weeks of gestation delivered uninfected, healthy infants. Attempts to demonstrate transplacental passage of Coxsackie B-3 or ECHO-11 viruses by use of the dual, recirculating, in vitro perfusion of an isolated placental lobule were unsuccessful even with a maternal virus input of 200,000 median tissue culture infectious doses. This suggests that transplacental passage of virus does not occur readily, and that most neonates of infected mothers will be unharmed. (AM J OasTET GVNECOL 1988;158:775-82.)
Key words: Enterovirus, transplacental passage, maternal virus infection, placental perfusion Clinical and laboratory studies of neonatal enterovirus infections have failed to define risk factors, sources of infection, and the biologic causes for increased neonatal susceptibility to these viruses. Because 80% to 90% of such infections occur in children 16 years old or younger, similar information about adults who are infected with enteroviruses is even more inadequate. The difference in enterovirus infection between adults and neonates is not understood, which adds to the difficulty in studying perinatal enterovirus infections. Analysis of several case reports of newborns with various enterovirus infections suggests that a transplacental mode of transmission is possible. I However, in only a rare instance was placental evidence of virus demonstrated." Practically all these reports were generated after the authors studied infected infants and then drew conclusions about pathogenesis. There are very few reports of clinically infected pregnant women who received neonatal follow-up." The present study was undertaken to determine the patterns of adult enterovirus infection and to study a group of pregnant women who had a known enterovirus infection before delivery. We also investigated transplacental passage of virus by use of the in vitro, dual-perfused human placental lobule. Polioviruses were eliminated from consideration in
From the Departments of Obstetrics-Gynecology, Microbiology, and Pathology, The University of Rochester School of Medicine and Dentistry. Supported in part by National Institutes of Health Grant No.
ES02774. Presented at the Sixth Annual Meeting ofthe American Gynecological and Obstetrical Society, Phoenix, Arizona, September 10-12,1987. Reprints not available.
this study of enterovirus infection because their control is nearly complete due to adequate immunization practices. Also, enterovirus 72 is now identified as hepatitis A" All enteroviruses are similar enough physically, chemically, and genetically that is reasonable to consider them together,"
Methods The records of the Monroe County, New York, diagnostic virology laboratory were reviewed for the period January 1976 through December 1985. Each enterovirus isolation was recorded, noting in particular the date of infection, age, site of isolation, clinical symptoms, and diagnosis of pregnancy. The specimens were obtained from symptomatic patients as they presented to their physicians. Because this is a retrospective analysis, all patients with positive specimens were included in this review. As a retrospective review, there is no representative sample from any particular group of individuals. No attempts were made to diagnose pregnancy, per se, in any female included in this survey. Only those women noted to be pregnant at the time of enterovirus isolation are included in the details discussed below. All clinical histories were obtained by a detailed chart review. None of the charts provided pathology detail about the placentas from the infected women who delivered. For purposes of the study, adults were defined as age 17 years or older. Clinical specimens were obtained by inoculating a virus transport medium with blood, cerebrospinal fluid, stool, vesicle fluid, or a swab of a mucous membrane. This medium or placental perfusion samples were inoculated variously onto monolayer cultures maintained with Eagle's minimal essential medium with or without fetal bovine serum depending on cell type used. Identification of isolates was performed by specific antisera 775
776 Amstay at al.
April 1988 Am J Obstet Gynecol
Table I. Enterovirus isolations, 1976-1985 Virus
All
Adults* (%)
Echovirus Coxsackie A Coxsackie B Enterovirus 71
560 68 190 10
16 (2.8%) 14 (20.6%) 21 (11%) 2 (20%)
Total
828t
-
53
*Age ~ 17 years. t Identified viruses (128 total and 21 adult isolates were not identified).
Table III. Distribution of adult enterovirus infections by age Age (yr)
No.
%
17-19 20-29 30-39 40-49 50-59 60-69 70 Unstated
8 42 16 3
10.8 56.8 21.5 4.0 1.4 1.4 1.4 2.7
Total
74
I I I
2
100
Table II. Distribution of adult enterovirus infections by quarter Quarter
No.
%
January-March April-June July-September October-Decem ber
0 6 54 14
0 8.1 73.0 18.9
Total
74*
100.0
*From a total of 957 isolates, adult infection comprised 7.7% of the total (34 men and 40 women).
in a plaque reduction assay. Details of these procedures have been published." Details of the placental perfusion techniques were described at the Fourth Annual Meeting of the American Gynecological and Obstetrical Society in 1985 and reported subsequently." In summary, the dual, recirculating in vitro perfusion of an isolated human placentallobule was modified extensively from the original work of Panigel" as reported in 1962 for long-term perfusion. The placentas were obtained from two women undergoing elective repeat cesarean sections who had uncomplicated prenatal courses. The mothers' serum samples were screened for antibody to the virus used for perfusion of each placenta. An initial2-hour control perfusion was done to establish all parameters before introducing 5 X 104 medium tissue culture infectious doses of Coxsackie B3 or 2 X 105 doses of ECHO II viruses. Details of the physical and physiologic parameters followed throughout the studies have been reported." The viruses chosen for inoculation were fresh clinical isolates in a less than sixth BGM tissue culture passage. Viral titers were measured in BGM cells after thawing and diluting in placental perfusion medium. Fetal and maternal arterial and/or venous samples of 1.5 ml were drawn after 2 hours of control observation and subsequently every 4 hours, and refrigerated for 12 hours at - 70 0 C for future virus titration. The biochemical tests were done on portions of the sample after freezing at - 20 0 C. These samples were used for viral
isolation, glucose, human chorionic gonadotropin, and lactate determinations. The presence or absence of viral particles and the morphologic changes and overall integrity of the samples were evaluated by electron microscopy at the end of each perfusion study. Results
For this survey, 957 individuals were found who were infected with various non polio enteroviruses. Data on the distribution among the four large enterovirus groups and the fraction that occurred in adults are presented in Table I. Whereas 68% of all individuals had an echovirus infection, only 2.8% of adults had such infections. However, 20% of adults had Coxsackie A virus infections. No single year in the IO-year survey had a disproportionate number of adult infections, which accounted for nearly 8% of all the enterovirus infections. In addition to the apparent random distribution of enteroviral infections by year, the distribution of the most prominent enterovirus infection in any given year was also random. Not only was a different enterovirus prevalent each of the 10 years, no enterovirus repeated as the most prevalent over the entire survey period. Historically, most enterovirus infections in temperate climates occur in the summer months. The same distribution was noted in the present survey (Table II). Nearly 92% of ali infections occurred in the second half of the year, and 73% occurred the third quarter. Adults accounted for 7.7% of the enterovirus infections, which were distributed in 34 men and 40 women. The age distribution of adults (Table III) illustrates that nearly 90% of infections occur in the child-bearing years between ages 17 and 40 years. Consequently, one would expect some adult enterovirus infections to occur in pregnant women. This was confirmed by noting that of the 40 female adults who developed an enterovirus infection, 14 were pregnant. Details of these 14 women are grouped by severity of disease (Table IV). The four pregnant women who
Enterovirus infection in pregnancy 777
Volume 158 Number 4
Table IV. Pregnant women with enterovirus infection Gestational age (wk)
Virus
Site of isolation
Meningitis S. S. L. P. M.R. K. A.
28 19 28 29
26 16 33* 29
ECHO 24 Cox B5 Cox B4 Cox B4
Throat, CSF Throat, CSF Throat, CSF CSF
M.A. D. T. A. S. P. D. A.A.
29 28 28 34 19
23 34 22 32 28
ECHO II Cox B4 Cox B4 ECHO 17 Cox B3
Throat Throat, rectum Throat, stool Stool Stool
M.K. K. C. C. D. B. D.t P. M.
22 26 28 18 26
37 16 39 29 35
Cox A9 Cox A16 Cox A? ECHO 6 ECHO 22
Cervix Oral lesion Vesicle Cervix Cervix
Fever, respiratory disease
HFM, minor respiratory disease, asymptomatic
ECHO, Echovirus; CSF, cerebrospinal fluid; Cox, coxsackievirus; HFM, hand-foot-and-mouth disease. *Delivered within 48 hours of admission. tPreterm labor, suppressed; delivered at 37 weeks' gestation.
presented with meningitis at 16 to 33 weeks of gestation had stiff necks, photophobias, headache, and fever >38.5° C. Patient M. R. delivered <48 hours after admission, producing a 2100 gm male with Apgar scores of 5 and 8 at 1 and 5 minutes, respectively. This infant was given 0.2 ml/kg of commercial pooled 'V-globulin. He and the other three babies from this group were all well at discharge (which occurred within 10 days). No virus was isolated from any of these infants. The next group of five pregnant women presented with fever and respiratory symptoms. None required hospital admission, and the various enteroviruses were isolated from throat and/or stool. The latest gestational age at which infection occurred in this group was 34 weeks. The third group of five pregnant women had either hand-foot-and-mouth disease, minor respiratory symptoms without fever, or were asymptomatic with positive viral isolations from the cervix. The 10 babies from the latter two groups of women were also healthy and well. A summary of all newborns delivered of these 14 infected women is shown in Table V. The mean (± SD) weight of 2980 ± 569 gm was not statistically different from the mean weight of 3171 ± 755 gm for all deliveries in the 10-year period surveyed for this study (chi-square analysis). There were no complications or neonatal difficulties attributed to any of these 14 mother's enterovirus infections. Table VI shows the physiologic status of the two placental perfusion studies. All parameters were considered normal and unchanged after perfusion with either virus and when compared with other perfusion studies.'? No virus could be detected in the fetal circulation
Table V. Neonatal outcome of 14 enterovirus-infected mothers 2980 (range 2100-3900) Mean weight (gm) 9 (range 8-10) Mean 5 min Apgar score Complications Transient tachypnea Hydrocele (due to ECHO 20 virus: Infant meningitis mother's cervix, age 4 mo ECHO-6 virus) Premature labor 2
after 6 hours of viral perfusion in the first study (Table VII). Despite a fourfold higher virus concentration and double the perfusion time in the second study, no virus could be detected in the fetal circulation. Electron inicroscopic studies of the first perfused placenta (Coxsackie B3 virus at 8 hours) indicated excellent subinicroscopic morphologic preservation of all cell types (trophoblast, fetal endothelial, and villus stromal cells). Large, distinct multilaminated or lamellar lipid bodies were noted in some syncytial trophoblastic cells (Fig. 1). Occasional fibrin thrombi were noted in fetal vessels, as were rare bacilliform bacteria. No viral particles were identified in fetal vascular spaces or within any cells (trophoblast, endothelial, stromal) after an extensive search at high magnification. Electron microscopic studies of the second perfused placenta likewise showed no evidence of intra- or extracellular viral particles. Morphologic preservation was only moderately good but similar to that of an adjacent nonperfused
778 Amstey et al.
April 1988 Am J Obstet Gynecol
Table VI. Physiologic status of virus-perfused human placental lobule Condition
Fetal pressure (mm Hg)
Fetal volume (mllmin)
hCG release (IV/min' kg)
Glucose utilization (umoll min . kg)
1-2 3-5 5-8
Control Coxsackie B3* Coxsackie B3
23 ± I 28 ± 3 34 ± 3
<2 <2 <2
88 ± 8 94 ± 10 84 ± 11
120 ± 8 114 ± 10 122 ± 6
1-2 3-6 7-12 12-15
Control ECHO 11* ECHO lit ECHO 1I:j:
<2 <2 <2 <2
51 58 47 63
Time (hr)
20 20.5 22 21
± ± ± ±
0 0.3 0 3
65 83 55 61
± 10 ± 16 ± 7 ± 12
± ± ± ±
12 20 16 6
Data are X ± SD. hCG, Human chorionic gonadotropin; ECHO, echovirus. *See Table VII for amount of virus inoculated into maternal circulation. t I x 10' mean tissue culture infectious doses after one complete maternal exchange transfusion. :j:5 x 103 mean tissue culture infectious doses after second complete maternal exchange transfusion.
Table VII. Perfusion of isolated placental lobule with enterovirus Study*
Virus
I
Coxsackie B3 ECHO-II
2
Virus titer inoculatedt
5
X
2
X
10' 10'
Maternal titer 4 hr after second exchange
Virus in fetal circulation
NA 5 x 103 :j:
ND ND
NA, Not applicable; ND, none detected. *Virus was perfused for 6 hours in the first study and 12 hours in the second study. t Fifty percent tissue culture infectious doses per milliliter of maternal perfusate; total volume :j:The titer at the conclusion of the experiment was 2 X 103 •
lobule taken as a control. No lamellar bodies, fibrin thrombi, or bacteria were noted. Comment
The epidemiologic observations made in this study are similar to the large enteroviral surveillance data published by the Centers for Disease Control." Although 7.7% of individuals in the current study were ~ 17 years old, the national survey found 13.5% were ~20 years old. However, both studies showed the wellknown seasonal variation in these infections. Unfortunately, the national survey had no breakdown for enterovirus infection during pregnancy. A survey of 654 healthy women at term failed to demonstrate any enterovirus on the day of delivery," indicating that enterovirus infections are not common at the end of pregnancy. Therefore, a group of 14 pregnant women with an enterovirus infection during pregnancy is a large group that may allow some meaningful statements about perinatal infection to be made. The present findings of no symptomatically infected neonates strengthens the suggestion that vertical transmission of nonpolio enteroviruses with resultant clinical disease also is not common. There are several reports that demonstrate in utero infection with these viruses," but the true incidence of such infection must be very low. In fact, after a search of the world's literature,
=
300 ml,
Modlin':' indicated that no more than 24 cases of echovirus infection could have occurred in utero. The relatively high incidence of neonatal infection, generally, is due to nosocomial infection in the nursery and not the result of mother-to-neonate infection. One anecdotal piece of information described in our clinical survey is the possible benefit from commercial ,,-globulin given to a neonate delivered of a mother currently ill from a serious enterovirus meningitis. It had been suggested that neonates born during such circumstances are at greater risk for enterovirus disease because of the lack of maternal antibodies available so soon after the onset of maternal illness. '2. 15 The antibody level for Coxsackie B4 virus in different lots of human serum globulin is high." All the remaining neonates in this study delivered long enough after maternal infection that it is possible to speculate that protective maternal antibody was available to prevent neonatal infection if it were to occur by any route of transmission. The mechanism of enterovirus transmission from mother to fetus when it does occur is not clear. One's first response is to suggest that transplacental passage of virus occurs; however, in only a few instances has any nonpolio enterovirus been isolated from placenta." Although one mouse study purported to show placental virus, the processing of the specimens could not avoid
Enterovirus infection in pregnancy 779
Volume 158 Number 4
Fig. 1. Placenta with Coxsackie B3 virus shows syncytiotrophoblast (ST), cytotrophoblast (CT). and fetal endotheluim (E). Note the excellent preservation of nuclei, the submicroscopic cytoplasmic organelles, microvilli, mitochondria, rough endoplasmic recticulum, polysomes, and Golgi apparatus. Numerous lamellar bodies are present in the syncytiotrophoblast. No intravascular or intracellular viral particles were noted. (Original magnification x 6000.)
cross-contamination from the uterus." The preliminary findings of the absence of virus in the fetal circulation or in microscopic surveillance of thin sections of the perfused placenta suggests strongly that Coxsackie B3 and ECHO II viruses will not infect or cross the placenta in 12 hours. This time frame is certainly long enough to establish placental infection and produce progeny virus in the placenta-if it were capable of doing SO.19 Future experiments will be needed to confirm this finding and to lengthen this perfusion time to exclude fully the time factor. The ultrastructural findings of lamellar bodies and fibrin thrombi in the first perfusion study are probably nonspecific and not related to the viral perfusion, because they were not seen in the second study. These two findings could be related to either preexisting changes and/or perfusion-related damage. Perhaps an alternative explanation for fetal infection in utero is transmembrane passage of virus from an infected cervix or vagina. In 1984 Galask et al." demonstrated the possibility of bacteria penetrating the amnion and chorion. There is no reason to believe that viruses could not do likewise. In fact, amnion cells are infected readily by enteroviruses and many other human viruses." It is not clear at this time what role maternal infection
from enteroviruses plays in the development of perinatal infection. However, we believe that it is safe to state at this time that most maternal nonpolio enteroviral infection will cause little or no harm to the fetus or neonate. In conclusion, adults account for nearly 8% of nonpolio enteroviral infection, the majority of whom will be in the child-bearing age group. Furthermore, most of these infections will occur in the third quarter of the year. Which non polio enterovirus is prevalent in any year is random, but most adult infections will be from coxsackieviruses. Pregnant women with serious or minor coxsackievirus and echovirus infections may be expected to have uncomplicated deliveries and healthy babies. Perfusion of the isolated placental lobule in vitro with large amounts of two different enteroviruses failed to demonstrate transplacental passage of these viruses. Finally, the lack of perinatal illness among a relatively large group of women with nonpolio enterovirus infection and the failure to demonstrate transplacental passage of these viruses under our conditions suggest that transplacental passage of nonpolio enteroviruses is probably uncommon. We thank Christine Mayer and Karen Jensen for their expert laboratory assistance.
780
Amstey et al.
REFERENCES I. Lake AM, Lauer BA, Clark JC, et al. Enterovirus infections in neonates. J Pediatr 1976;89:787-91. 2. Benirschke K, Pendleton M. Coxsackie virus infection. Obstet Gynecol 1958; 12:305-9. 3. Reyes MP, Salenski D, Smith F, et al. Coxsackieviruspositive cervices in women with febrile illnesses during the third trimester in pregnancy. AM J OBSTET GYNECOL 1986; 133: 159-61. 4. MeinickJ. Classification of hepatitis A virus as enterovirus type 72 and hepatitis B virus as hepadnavirus type I. Intervirology 1982; 18: 105-6. 5. Huang A, Modlin J. Pathogenesis and immunity in enteroviral infections. In: Madell G, Douglas R, BennettJ, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley and Sons, 1985:802-3. 6. Menegus M, Hollick G. Increased efficiency of group B Coxsackie virus isolation from clinical specimens by use of BGM cells. J Clin Microbiol 1982;15:945-8. 7. Miller R, Wier P, Maulik D, et al. Human placenta in vitro: characterization during 12h of dual perfusion. Contrib GynecolObstet 1985;13:77-84. 8. Panigel M. Placental perfusion experiments. AMJ OBSTET GYNECOL 1962;84: 1664-72. 9. Wier P, Miller R. Oxygen transfer as an indicator of perfusion variability in the isolated human placental lobule. Contrib Gynecol Obstet 1985;13:127-31. 10. Wier P, Miller R. The pharmokinetics of cadmium in the dually perfused human placenta. Trophoblast Res 1987;2:357-66. II. Enterovirus surveillance: Summary 1970-1979. Atlanta: Centers for Disease Control, 1981. 12. Dagan R,JenistaJ, Menegus M. Clinical, epidemiological, and laboratory aspects of enterovirus infection in young infants. In: de la Maza L, Peterson E, eds. Medical virology IV. Hillsdale, NJ: L Eribaum Assoc, 1985: 123-51. 13. JenistaJ, Menegus M. Enterovirus: Coxsackie, ECHO and poliovirus. In: Amstey M, ed. Virus infection in pregnancy. Orlando: Grune & Stratton, 1984:1-17. 14. Modlin J. Perinatal echovirus infection: insights from a literature review of 61 cases of serious infection and 16 outbreaks in nurseries. Rev Infect Dis 1986;8:918-26. 15. Modlin J, Polk B, Horton R, et al. Perinatal echovirus infection: risk of transmission during a community outbreak. N EnglJ Med 1981;305:368-71. 16. Dagan R, Prather S, Powell K, et al. Neutralizing antibodies to non-polio er teroviruses in human immune serum globulin. Pediatr .nfect Dis 1983;2:454-6. 17. Brightman V, Scott T, 'Testphal M, et al. An outbreak of Coxsackie B5 virus infec.ion in a newborn nursery. J Pediatr 1966;69: 179-92. 18. Modlin J, Crumpacker C. Coxsackievirus B infection in pregnant mice and transplacental infection of the fetus. Infect Immun 1982;37:222-6. 19. Huang A, Modlin J. RNA viruses: Picornaviridae. In: Mandell G, Douglas R, BennettJ, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley and Sons, 1985:802. 20. Galask R, Varner M, Petzold R, et al. Bacterial attachment to the chorioamniotic membranes. AM J OBSTET GYNECOL 1984; 148:915-28. 21. Horstmann D. The picornavirus group. In: Horsfall F Jr, Tamm I, eds. Viral and rickettsial infections of man. 4th ed. Philadelphia: JB Lippincott, 1965:425.
Editors' note: This manuscript was revised after these discus-
sions were presented.
Discussion
L. SWEET, San Francisco, California. Enteroviruses are small ribonucleic acid viruses and are DR. RICHARD
April 1988 Am J ObsrerGynecol
classified into five groups: polio viruses, Coxsackie A viruses, Coxsackie B viruses, echoviruses, and enteroviruses 68-72 (enterovirus 72 is Hepatitis A virus). Enteroviruses infect humans primarily as the result of ingestion of fecally contaminated material. The ingested virus implants and replicates in the lymphoid tissue of the gastrointestinal tract. It then spreads to the reticuloendothelial system, resulting in a "minor viremia" that is transient and not usually detectable. The majority of infections are subclinical, and virus replication ceases at this point. In a minority of infected persons, further replication occurs in the reticuloendothelial system and the virus disseminates to secondary organs such as skin, heart, liver, and central nervous system, resulting in a "major viremia." It is at this time that transplacental spread of enteroviruses may occur. Unfortunately, this phase most often presents clinically as a nonspecific febrile illness lasting 2 to 5 days. Less commonly, patients present with a variety of respriatory syndromes, aseptic meningitis, gastrointestinal disease, or hand-foot-and-mouth disease. Although most enterovirus infections are in children, Dr. Amstey notes that roughly 10% of reported nonpolio enteroviruses occur in adults, most being of reproductive age.' Not surprising, sporadic and epidemic infections with non polio enteroviruses have been reported in pregnant women and their newborns." There is no good evidence that non polio enteroviruses are causes of abortion or stillbirth. Two large studies have demonstrated an increased incidence of coxsackievirus seroconversions in mothers of children with congenital anomalies. Among Finnish women Coxsackie B5 virus infection was more common,' and in the United States significant associations of Coxsackie B2 and B4 viruses with urogenital anomalies, Coxsackie A9 virus with gastrointestinal defects, and Coxsackie B3 and B4 viruses with cardiovascular lesions were reported." No such association has been demonstrated for the echoviruses. It has been suggested that neonates are uniquely susceptible to enterovirus infection. The enteroviruses are capable of causing severe disease and death when infection occurs within the first 10 to 14 days of life. Although transplacental transmission of virus can occur throughout gestation, most neonates with evidence of in utero acquisition of infection appear to have become infected in 'the week before delivery. However, only 11% of neonatal echovirus diseases" and 22% of neonatal group B coxsackievirus diseases' have been demonstrated to occur as a result of antepartum intrauterine transmission. Most cases present several days after birth and are believed to represent intrapartum or nocosomial acquisition of enteroviruses, Thus the situation seems analogous to the varicella experience, in which maternal infection that occurs in close proximity to delivery is associated with a risk of severe neonatal infection, probably due to a lack of transplacentally acquired maternal antibody. Both the incidence of neonatal non polio enterovirus infection and the risk of vertical transmission of maternal enterovirus infection are unknown.
Key words: Enterovirus, transplacental passage, maternal virus infection, placental perfusion Clinical and laboratory studies of neonatal enterovirus infections have failed to define risk factors, sources of infection, and the biologic causes for increased neonatal susceptibility to these viruses. Because 80% to 90% of such infections occur in children 16 years old or younger, similar information about adults who are infected with enteroviruses is even more inadequate. The difference in enterovirus infection between adults and neonates is not understood, which adds to the difficulty in studying perinatal enterovirus infections. Analysis of several case reports of newborns with various enterovirus infections suggests that a transplacental mode of transmission is possible. I However, in only a rare instance was placental evidence of virus demonstrated." Practically all these reports were generated after the authors studied infected infants and then drew conclusions about pathogenesis. There are very few reports of clinically infected pregnant women who received neonatal follow-up." The present study was undertaken to determine the patterns of adult enterovirus infection and to study a group of pregnant women who had a known enterovirus infection before delivery. We also investigated transplacental passage of virus by use of the in vitro, dual-perfused human placental lobule. Polioviruses were eliminated from consideration in
From the Departments of Obstetrics-Gynecology, Microbiology, and Pathology, The University of Rochester School of Medicine and Dentistry. Supported in part by National Institutes of Health Grant No.
ES02774. Presented at the Sixth Annual Meeting ofthe American Gynecological and Obstetrical Society, Phoenix, Arizona, September 10-12,1987. Reprints not available.
this study of enterovirus infection because their control is nearly complete due to adequate immunization practices. Also, enterovirus 72 is now identified as hepatitis A" All enteroviruses are similar enough physically, chemically, and genetically that is reasonable to consider them together,"
Methods The records of the Monroe County, New York, diagnostic virology laboratory were reviewed for the period January 1976 through December 1985. Each enterovirus isolation was recorded, noting in particular the date of infection, age, site of isolation, clinical symptoms, and diagnosis of pregnancy. The specimens were obtained from symptomatic patients as they presented to their physicians. Because this is a retrospective analysis, all patients with positive specimens were included in this review. As a retrospective review, there is no representative sample from any particular group of individuals. No attempts were made to diagnose pregnancy, per se, in any female included in this survey. Only those women noted to be pregnant at the time of enterovirus isolation are included in the details discussed below. All clinical histories were obtained by a detailed chart review. None of the charts provided pathology detail about the placentas from the infected women who delivered. For purposes of the study, adults were defined as age 17 years or older. Clinical specimens were obtained by inoculating a virus transport medium with blood, cerebrospinal fluid, stool, vesicle fluid, or a swab of a mucous membrane. This medium or placental perfusion samples were inoculated variously onto monolayer cultures maintained with Eagle's minimal essential medium with or without fetal bovine serum depending on cell type used. Identification of isolates was performed by specific antisera 775
776 Amstay at al.
April 1988 Am J Obstet Gynecol
Table I. Enterovirus isolations, 1976-1985 Virus
All
Adults* (%)
Echovirus Coxsackie A Coxsackie B Enterovirus 71
560 68 190 10
16 (2.8%) 14 (20.6%) 21 (11%) 2 (20%)
Total
828t
-
53
*Age ~ 17 years. t Identified viruses (128 total and 21 adult isolates were not identified).
Table III. Distribution of adult enterovirus infections by age Age (yr)
No.
%
17-19 20-29 30-39 40-49 50-59 60-69 70 Unstated
8 42 16 3
10.8 56.8 21.5 4.0 1.4 1.4 1.4 2.7
Total
74
I I I
2
100
Table II. Distribution of adult enterovirus infections by quarter Quarter
No.
%
January-March April-June July-September October-Decem ber
0 6 54 14
0 8.1 73.0 18.9
Total
74*
100.0
*From a total of 957 isolates, adult infection comprised 7.7% of the total (34 men and 40 women).
in a plaque reduction assay. Details of these procedures have been published." Details of the placental perfusion techniques were described at the Fourth Annual Meeting of the American Gynecological and Obstetrical Society in 1985 and reported subsequently." In summary, the dual, recirculating in vitro perfusion of an isolated human placentallobule was modified extensively from the original work of Panigel" as reported in 1962 for long-term perfusion. The placentas were obtained from two women undergoing elective repeat cesarean sections who had uncomplicated prenatal courses. The mothers' serum samples were screened for antibody to the virus used for perfusion of each placenta. An initial2-hour control perfusion was done to establish all parameters before introducing 5 X 104 medium tissue culture infectious doses of Coxsackie B3 or 2 X 105 doses of ECHO II viruses. Details of the physical and physiologic parameters followed throughout the studies have been reported." The viruses chosen for inoculation were fresh clinical isolates in a less than sixth BGM tissue culture passage. Viral titers were measured in BGM cells after thawing and diluting in placental perfusion medium. Fetal and maternal arterial and/or venous samples of 1.5 ml were drawn after 2 hours of control observation and subsequently every 4 hours, and refrigerated for 12 hours at - 70 0 C for future virus titration. The biochemical tests were done on portions of the sample after freezing at - 20 0 C. These samples were used for viral
isolation, glucose, human chorionic gonadotropin, and lactate determinations. The presence or absence of viral particles and the morphologic changes and overall integrity of the samples were evaluated by electron microscopy at the end of each perfusion study. Results
For this survey, 957 individuals were found who were infected with various non polio enteroviruses. Data on the distribution among the four large enterovirus groups and the fraction that occurred in adults are presented in Table I. Whereas 68% of all individuals had an echovirus infection, only 2.8% of adults had such infections. However, 20% of adults had Coxsackie A virus infections. No single year in the IO-year survey had a disproportionate number of adult infections, which accounted for nearly 8% of all the enterovirus infections. In addition to the apparent random distribution of enteroviral infections by year, the distribution of the most prominent enterovirus infection in any given year was also random. Not only was a different enterovirus prevalent each of the 10 years, no enterovirus repeated as the most prevalent over the entire survey period. Historically, most enterovirus infections in temperate climates occur in the summer months. The same distribution was noted in the present survey (Table II). Nearly 92% of ali infections occurred in the second half of the year, and 73% occurred the third quarter. Adults accounted for 7.7% of the enterovirus infections, which were distributed in 34 men and 40 women. The age distribution of adults (Table III) illustrates that nearly 90% of infections occur in the child-bearing years between ages 17 and 40 years. Consequently, one would expect some adult enterovirus infections to occur in pregnant women. This was confirmed by noting that of the 40 female adults who developed an enterovirus infection, 14 were pregnant. Details of these 14 women are grouped by severity of disease (Table IV). The four pregnant women who
Enterovirus infection in pregnancy 777
Volume 158 Number 4
Table IV. Pregnant women with enterovirus infection Gestational age (wk)
Virus
Site of isolation
Meningitis S. S. L. P. M.R. K. A.
28 19 28 29
26 16 33* 29
ECHO 24 Cox B5 Cox B4 Cox B4
Throat, CSF Throat, CSF Throat, CSF CSF
M.A. D. T. A. S. P. D. A.A.
29 28 28 34 19
23 34 22 32 28
ECHO II Cox B4 Cox B4 ECHO 17 Cox B3
Throat Throat, rectum Throat, stool Stool Stool
M.K. K. C. C. D. B. D.t P. M.
22 26 28 18 26
37 16 39 29 35
Cox A9 Cox A16 Cox A? ECHO 6 ECHO 22
Cervix Oral lesion Vesicle Cervix Cervix
Fever, respiratory disease
HFM, minor respiratory disease, asymptomatic
ECHO, Echovirus; CSF, cerebrospinal fluid; Cox, coxsackievirus; HFM, hand-foot-and-mouth disease. *Delivered within 48 hours of admission. tPreterm labor, suppressed; delivered at 37 weeks' gestation.
presented with meningitis at 16 to 33 weeks of gestation had stiff necks, photophobias, headache, and fever >38.5° C. Patient M. R. delivered <48 hours after admission, producing a 2100 gm male with Apgar scores of 5 and 8 at 1 and 5 minutes, respectively. This infant was given 0.2 ml/kg of commercial pooled 'V-globulin. He and the other three babies from this group were all well at discharge (which occurred within 10 days). No virus was isolated from any of these infants. The next group of five pregnant women presented with fever and respiratory symptoms. None required hospital admission, and the various enteroviruses were isolated from throat and/or stool. The latest gestational age at which infection occurred in this group was 34 weeks. The third group of five pregnant women had either hand-foot-and-mouth disease, minor respiratory symptoms without fever, or were asymptomatic with positive viral isolations from the cervix. The 10 babies from the latter two groups of women were also healthy and well. A summary of all newborns delivered of these 14 infected women is shown in Table V. The mean (± SD) weight of 2980 ± 569 gm was not statistically different from the mean weight of 3171 ± 755 gm for all deliveries in the 10-year period surveyed for this study (chi-square analysis). There were no complications or neonatal difficulties attributed to any of these 14 mother's enterovirus infections. Table VI shows the physiologic status of the two placental perfusion studies. All parameters were considered normal and unchanged after perfusion with either virus and when compared with other perfusion studies.'? No virus could be detected in the fetal circulation
Table V. Neonatal outcome of 14 enterovirus-infected mothers 2980 (range 2100-3900) Mean weight (gm) 9 (range 8-10) Mean 5 min Apgar score Complications Transient tachypnea Hydrocele (due to ECHO 20 virus: Infant meningitis mother's cervix, age 4 mo ECHO-6 virus) Premature labor 2
after 6 hours of viral perfusion in the first study (Table VII). Despite a fourfold higher virus concentration and double the perfusion time in the second study, no virus could be detected in the fetal circulation. Electron inicroscopic studies of the first perfused placenta (Coxsackie B3 virus at 8 hours) indicated excellent subinicroscopic morphologic preservation of all cell types (trophoblast, fetal endothelial, and villus stromal cells). Large, distinct multilaminated or lamellar lipid bodies were noted in some syncytial trophoblastic cells (Fig. 1). Occasional fibrin thrombi were noted in fetal vessels, as were rare bacilliform bacteria. No viral particles were identified in fetal vascular spaces or within any cells (trophoblast, endothelial, stromal) after an extensive search at high magnification. Electron microscopic studies of the second perfused placenta likewise showed no evidence of intra- or extracellular viral particles. Morphologic preservation was only moderately good but similar to that of an adjacent nonperfused
778 Amstey et al.
April 1988 Am J Obstet Gynecol
Table VI. Physiologic status of virus-perfused human placental lobule Condition
Fetal pressure (mm Hg)
Fetal volume (mllmin)
hCG release (IV/min' kg)
Glucose utilization (umoll min . kg)
1-2 3-5 5-8
Control Coxsackie B3* Coxsackie B3
23 ± I 28 ± 3 34 ± 3
<2 <2 <2
88 ± 8 94 ± 10 84 ± 11
120 ± 8 114 ± 10 122 ± 6
1-2 3-6 7-12 12-15
Control ECHO 11* ECHO lit ECHO 1I:j:
<2 <2 <2 <2
51 58 47 63
Time (hr)
20 20.5 22 21
± ± ± ±
0 0.3 0 3
65 83 55 61
± 10 ± 16 ± 7 ± 12
± ± ± ±
12 20 16 6
Data are X ± SD. hCG, Human chorionic gonadotropin; ECHO, echovirus. *See Table VII for amount of virus inoculated into maternal circulation. t I x 10' mean tissue culture infectious doses after one complete maternal exchange transfusion. :j:5 x 103 mean tissue culture infectious doses after second complete maternal exchange transfusion.
Table VII. Perfusion of isolated placental lobule with enterovirus Study*
Virus
I
Coxsackie B3 ECHO-II
2
Virus titer inoculatedt
5
X
2
X
10' 10'
Maternal titer 4 hr after second exchange
Virus in fetal circulation
NA 5 x 103 :j:
ND ND
NA, Not applicable; ND, none detected. *Virus was perfused for 6 hours in the first study and 12 hours in the second study. t Fifty percent tissue culture infectious doses per milliliter of maternal perfusate; total volume :j:The titer at the conclusion of the experiment was 2 X 103 •
lobule taken as a control. No lamellar bodies, fibrin thrombi, or bacteria were noted. Comment
The epidemiologic observations made in this study are similar to the large enteroviral surveillance data published by the Centers for Disease Control." Although 7.7% of individuals in the current study were ~ 17 years old, the national survey found 13.5% were ~20 years old. However, both studies showed the wellknown seasonal variation in these infections. Unfortunately, the national survey had no breakdown for enterovirus infection during pregnancy. A survey of 654 healthy women at term failed to demonstrate any enterovirus on the day of delivery," indicating that enterovirus infections are not common at the end of pregnancy. Therefore, a group of 14 pregnant women with an enterovirus infection during pregnancy is a large group that may allow some meaningful statements about perinatal infection to be made. The present findings of no symptomatically infected neonates strengthens the suggestion that vertical transmission of nonpolio enteroviruses with resultant clinical disease also is not common. There are several reports that demonstrate in utero infection with these viruses," but the true incidence of such infection must be very low. In fact, after a search of the world's literature,
=
300 ml,
Modlin':' indicated that no more than 24 cases of echovirus infection could have occurred in utero. The relatively high incidence of neonatal infection, generally, is due to nosocomial infection in the nursery and not the result of mother-to-neonate infection. One anecdotal piece of information described in our clinical survey is the possible benefit from commercial ,,-globulin given to a neonate delivered of a mother currently ill from a serious enterovirus meningitis. It had been suggested that neonates born during such circumstances are at greater risk for enterovirus disease because of the lack of maternal antibodies available so soon after the onset of maternal illness. '2. 15 The antibody level for Coxsackie B4 virus in different lots of human serum globulin is high." All the remaining neonates in this study delivered long enough after maternal infection that it is possible to speculate that protective maternal antibody was available to prevent neonatal infection if it were to occur by any route of transmission. The mechanism of enterovirus transmission from mother to fetus when it does occur is not clear. One's first response is to suggest that transplacental passage of virus occurs; however, in only a few instances has any nonpolio enterovirus been isolated from placenta." Although one mouse study purported to show placental virus, the processing of the specimens could not avoid
Enterovirus infection in pregnancy 779
Volume 158 Number 4
Fig. 1. Placenta with Coxsackie B3 virus shows syncytiotrophoblast (ST), cytotrophoblast (CT). and fetal endotheluim (E). Note the excellent preservation of nuclei, the submicroscopic cytoplasmic organelles, microvilli, mitochondria, rough endoplasmic recticulum, polysomes, and Golgi apparatus. Numerous lamellar bodies are present in the syncytiotrophoblast. No intravascular or intracellular viral particles were noted. (Original magnification x 6000.)
cross-contamination from the uterus." The preliminary findings of the absence of virus in the fetal circulation or in microscopic surveillance of thin sections of the perfused placenta suggests strongly that Coxsackie B3 and ECHO II viruses will not infect or cross the placenta in 12 hours. This time frame is certainly long enough to establish placental infection and produce progeny virus in the placenta-if it were capable of doing SO.19 Future experiments will be needed to confirm this finding and to lengthen this perfusion time to exclude fully the time factor. The ultrastructural findings of lamellar bodies and fibrin thrombi in the first perfusion study are probably nonspecific and not related to the viral perfusion, because they were not seen in the second study. These two findings could be related to either preexisting changes and/or perfusion-related damage. Perhaps an alternative explanation for fetal infection in utero is transmembrane passage of virus from an infected cervix or vagina. In 1984 Galask et al." demonstrated the possibility of bacteria penetrating the amnion and chorion. There is no reason to believe that viruses could not do likewise. In fact, amnion cells are infected readily by enteroviruses and many other human viruses." It is not clear at this time what role maternal infection
from enteroviruses plays in the development of perinatal infection. However, we believe that it is safe to state at this time that most maternal nonpolio enteroviral infection will cause little or no harm to the fetus or neonate. In conclusion, adults account for nearly 8% of nonpolio enteroviral infection, the majority of whom will be in the child-bearing age group. Furthermore, most of these infections will occur in the third quarter of the year. Which non polio enterovirus is prevalent in any year is random, but most adult infections will be from coxsackieviruses. Pregnant women with serious or minor coxsackievirus and echovirus infections may be expected to have uncomplicated deliveries and healthy babies. Perfusion of the isolated placental lobule in vitro with large amounts of two different enteroviruses failed to demonstrate transplacental passage of these viruses. Finally, the lack of perinatal illness among a relatively large group of women with nonpolio enterovirus infection and the failure to demonstrate transplacental passage of these viruses under our conditions suggest that transplacental passage of nonpolio enteroviruses is probably uncommon. We thank Christine Mayer and Karen Jensen for their expert laboratory assistance.
780
Amstey et al.
REFERENCES I. Lake AM, Lauer BA, Clark JC, et al. Enterovirus infections in neonates. J Pediatr 1976;89:787-91. 2. Benirschke K, Pendleton M. Coxsackie virus infection. Obstet Gynecol 1958; 12:305-9. 3. Reyes MP, Salenski D, Smith F, et al. Coxsackieviruspositive cervices in women with febrile illnesses during the third trimester in pregnancy. AM J OBSTET GYNECOL 1986; 133: 159-61. 4. MeinickJ. Classification of hepatitis A virus as enterovirus type 72 and hepatitis B virus as hepadnavirus type I. Intervirology 1982; 18: 105-6. 5. Huang A, Modlin J. Pathogenesis and immunity in enteroviral infections. In: Madell G, Douglas R, BennettJ, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley and Sons, 1985:802-3. 6. Menegus M, Hollick G. Increased efficiency of group B Coxsackie virus isolation from clinical specimens by use of BGM cells. J Clin Microbiol 1982;15:945-8. 7. Miller R, Wier P, Maulik D, et al. Human placenta in vitro: characterization during 12h of dual perfusion. Contrib GynecolObstet 1985;13:77-84. 8. Panigel M. Placental perfusion experiments. AMJ OBSTET GYNECOL 1962;84: 1664-72. 9. Wier P, Miller R. Oxygen transfer as an indicator of perfusion variability in the isolated human placental lobule. Contrib Gynecol Obstet 1985;13:127-31. 10. Wier P, Miller R. The pharmokinetics of cadmium in the dually perfused human placenta. Trophoblast Res 1987;2:357-66. II. Enterovirus surveillance: Summary 1970-1979. Atlanta: Centers for Disease Control, 1981. 12. Dagan R,JenistaJ, Menegus M. Clinical, epidemiological, and laboratory aspects of enterovirus infection in young infants. In: de la Maza L, Peterson E, eds. Medical virology IV. Hillsdale, NJ: L Eribaum Assoc, 1985: 123-51. 13. JenistaJ, Menegus M. Enterovirus: Coxsackie, ECHO and poliovirus. In: Amstey M, ed. Virus infection in pregnancy. Orlando: Grune & Stratton, 1984:1-17. 14. Modlin J. Perinatal echovirus infection: insights from a literature review of 61 cases of serious infection and 16 outbreaks in nurseries. Rev Infect Dis 1986;8:918-26. 15. Modlin J, Polk B, Horton R, et al. Perinatal echovirus infection: risk of transmission during a community outbreak. N EnglJ Med 1981;305:368-71. 16. Dagan R, Prather S, Powell K, et al. Neutralizing antibodies to non-polio er teroviruses in human immune serum globulin. Pediatr .nfect Dis 1983;2:454-6. 17. Brightman V, Scott T, 'Testphal M, et al. An outbreak of Coxsackie B5 virus infec.ion in a newborn nursery. J Pediatr 1966;69: 179-92. 18. Modlin J, Crumpacker C. Coxsackievirus B infection in pregnant mice and transplacental infection of the fetus. Infect Immun 1982;37:222-6. 19. Huang A, Modlin J. RNA viruses: Picornaviridae. In: Mandell G, Douglas R, BennettJ, eds. Principles and practice of infectious diseases. 2nd ed. New York: John Wiley and Sons, 1985:802. 20. Galask R, Varner M, Petzold R, et al. Bacterial attachment to the chorioamniotic membranes. AM J OBSTET GYNECOL 1984; 148:915-28. 21. Horstmann D. The picornavirus group. In: Horsfall F Jr, Tamm I, eds. Viral and rickettsial infections of man. 4th ed. Philadelphia: JB Lippincott, 1965:425.
Editors' note: This manuscript was revised after these discus-
sions were presented.
Discussion
L. SWEET, San Francisco, California. Enteroviruses are small ribonucleic acid viruses and are DR. RICHARD
April 1988 Am J ObsrerGynecol
classified into five groups: polio viruses, Coxsackie A viruses, Coxsackie B viruses, echoviruses, and enteroviruses 68-72 (enterovirus 72 is Hepatitis A virus). Enteroviruses infect humans primarily as the result of ingestion of fecally contaminated material. The ingested virus implants and replicates in the lymphoid tissue of the gastrointestinal tract. It then spreads to the reticuloendothelial system, resulting in a "minor viremia" that is transient and not usually detectable. The majority of infections are subclinical, and virus replication ceases at this point. In a minority of infected persons, further replication occurs in the reticuloendothelial system and the virus disseminates to secondary organs such as skin, heart, liver, and central nervous system, resulting in a "major viremia." It is at this time that transplacental spread of enteroviruses may occur. Unfortunately, this phase most often presents clinically as a nonspecific febrile illness lasting 2 to 5 days. Less commonly, patients present with a variety of respriatory syndromes, aseptic meningitis, gastrointestinal disease, or hand-foot-and-mouth disease. Although most enterovirus infections are in children, Dr. Amstey notes that roughly 10% of reported nonpolio enteroviruses occur in adults, most being of reproductive age.' Not surprising, sporadic and epidemic infections with non polio enteroviruses have been reported in pregnant women and their newborns." There is no good evidence that non polio enteroviruses are causes of abortion or stillbirth. Two large studies have demonstrated an increased incidence of coxsackievirus seroconversions in mothers of children with congenital anomalies. Among Finnish women Coxsackie B5 virus infection was more common,' and in the United States significant associations of Coxsackie B2 and B4 viruses with urogenital anomalies, Coxsackie A9 virus with gastrointestinal defects, and Coxsackie B3 and B4 viruses with cardiovascular lesions were reported." No such association has been demonstrated for the echoviruses. It has been suggested that neonates are uniquely susceptible to enterovirus infection. The enteroviruses are capable of causing severe disease and death when infection occurs within the first 10 to 14 days of life. Although transplacental transmission of virus can occur throughout gestation, most neonates with evidence of in utero acquisition of infection appear to have become infected in 'the week before delivery. However, only 11% of neonatal echovirus diseases" and 22% of neonatal group B coxsackievirus diseases' have been demonstrated to occur as a result of antepartum intrauterine transmission. Most cases present several days after birth and are believed to represent intrapartum or nocosomial acquisition of enteroviruses, Thus the situation seems analogous to the varicella experience, in which maternal infection that occurs in close proximity to delivery is associated with a risk of severe neonatal infection, probably due to a lack of transplacentally acquired maternal antibody. Both the incidence of neonatal non polio enterovirus infection and the risk of vertical transmission of maternal enterovirus infection are unknown.