Martine Gagnon,J1 MD; Raymond Chicoine,II MD; Marc Boucher,** MD; Jacques Guimond,tt MD; ..... Laurent Berthiaume for performing the electron micro-.
CLINICAL AND COMMUNITY STUDIES a
ETUDES CLINIQUES ET COMMUNAUTAIRES
Intrauterine diagnosis of cytomegalovirus and rubella infections by amniocentesis Ranko Skvorc-Ranko,* MD; Henri Lavoie,t MD; Philippe St-Denis,4 MD; Robert Villeneuve,§ MD; Martine Gagnon,J1 MD; Raymond Chicoine,II MD; Marc Boucher,** MD; Jacques Guimond,tt MD; Yvon Dontigny,:# MD Objective: To establish a correlation between the presence of cytomegalovirus (CMV) or rubella virus in amniotic fluid obtained through amniocentesis and fetal infection. Design: Case series. Setting: Five hospitals in the Montreal region. Virology testing was done at the Virology Research Centre, Institut Armand-Frappier, Laval, Que. Patients: Thirteen pregnant women infected with CMV, 3 with rubella, their 15 babies and 2 fetuses. Twelve of the women with CMV infection were recruited from a prospective study of CMV infection in pregnancy. Infection in the other women was detected through routine laboratory diagnostic testing. Intervention: Amniotic fluid samples were cultured for CMV and rubella virus. Congenital infection of the neonates was established through isolation of either virus from pharyngeal mucus and urine specimens collected during the first 3 days of life or from fetal tissue if the pregnancy was terminated. Main results: CMV was cultured from the amniotic fluid of three of the CMV-infected women and from the pharyngeal mucus and urine specimens of their infants. Of the three women with rubella the amniotic fluid of one (who had a twin pregnancy) was positive for rubella virus. After the in-utero death of one fetus she underwent a therapeutic abortion of both. Examination of fetal tissue indicated that both fetuses had been infected with rubella virus. Each of the two other women with rubella gave birth to an uninfected, healthy infant. Conclusions: We found a strong correlation between the isolation of CMV or rubella virus from the amniotic fluid and the presence of congenital infection. This suggests that amniocentesis used to detect the presence of a virus is a useful method for the diagnosis of fetal infection.
Objectif: Etablir un lien entre l'infection foetale et la presence du cytomegalovirus (CMV) ou du virus de la rubeole dans le liquide amniotique preleve par amniocentese. Conception: Etude de cas. Contexte: Cinq h6pitaux de la region de Montreal. Les analyses virologiques ont ete effectuees a l'Institut Armand-Frappier de Laval (Que.). Patients: Treize femmes enceintes souffrant d'infection a CMV, 3 atteintes de rubeole, leurs 15 bebes et 2 foetus. Douze des femmes atteintes d'ure infection a CMV ont et recrutees d'une etude sur l'infection a CMV en cours de grossesse. L'infection chez les autres femmes a ete identifiee a la suite d'analyses de diagnostic de routine. From the *Virology Research Centre, Institut Armand-Frappier, Laval, Que.; the departments of tObstetrics and Gynecology and tPediatrics, H6pital Charles Le Moyne, Greenfield Park, Que.; the §Department of Obstetrics and Gynecology, Centre hospitalier Fleury, Montreal, Que.; the departments of 1Obstetrics and Gynecology and I/Pediatrics, H6pital Maisonneuve-Rosemont, Montreal, Que.; the departments of **Obstetrics and Gynecology and ttPediatrics, Hopital Sainte-Justine, Montreal, Que.; and the *$Department of Obstetrics and Gynecology, Centre hospitalier Sainte-Marie, Trois-Rivieres, Que.
Reprint requests to: Dr. Ranko Skvorc-Ranko, Institut Armand-Frappier, PO Box 100, Laval, PQ H7N 4Z3 -
For prescribing information see page 731
CAN MED ASSOC J 1991; 145 (6)
649
Intervention: On a effectue des cultures de specimens de liquide amniotique pour y depister le CMV et le virus de la rubeole. On a etabli qu'il y avait infection congenitale des nouveau-nes en isolant l'un ou I'autre virus a partir de frottis pharyngiens et de specimens d'urine preleves au cours des 3 premieres journees de la vie, ou du tissu foetal dans les cas ou l'on a mis fin a la grossesse. Principaux resultats: On a effectue des cultures de CMV a partir du liquide amniotique de trois des femmes infectees par le CMV et de frottis pharyngiens et de specimens d'urine de leurs nouveau-nes. Le liquide amniotique d'une des trois femmes atteintes de rubeole (qui portait des jumeaux) contenait le virus de la rubeole. Apres la mort in-utero d'un foetus, elle a subi un avortement therapeutique des deux. L'analyse du tissu foetal a revele que les deux foetus avaient e infectes par le virus de la rubeole. Les deux autres femmes atteintes du virus de la rubeole ont donne naissance a des nouveau-nes non infectes et en sante. Conclusions: Nous avons constate un lien solide entre l'isolement du CMV ou du virus de la rubeole dans le liquide amniotique et la presence d'infection congenitale, ce qui laisse entendre que l'amniocentese utilisee pour depister la presence d'un virus est une methode utile qui aide a diagnostiquer une infection foetale.
ransabdominal amniocentesis is a recognized prenatal procedure that allows diagnosis of certain genetic diseases at an early stage to permit termination of high-risk pregnancies. Cytomegalovirus (CMV) and rubella virus can be transmitted to an embryo or fetus by a woman infected shortly before or during her pregnancy. If the embryo or fetus becomes infected early in the pregnancy - particularly with rubella in the first trimester - there may be serious birth defects.'-"O Because pregnant women infected with either of these viruses are highly anxious about whether they will have a healthy infant it would be desirable to determine through amniocentesis whether the fetus has become infected. There have been only a few reports of the use of amniocentesis to diagnose infection by CMVI 1-8 and rubella virus'9 in the fetus. In a previous study20 CMV was identified in the amniotic fluid of a woman who had transmitted the infection to her fetus. The purpose of this study was to establish a correlation between the presence of CMV or rubella virus in amniotic fluid and infection in the fetus. T
The other woman with CMV infection was identified through a request by her physician for diagnostic analysis at the Virology Research Centre, Institut Armand-Frappier, Laval, Que. The three women with rubella, two at the Centre hospitalier Sainte-Marie, Trois-Rivieres, Que., and one at H6pital Maisonneuve-Rosemont, were also identified through routine testing at the Virology Research Centre.
Determination of type of infection Primary maternal infection was defined as seroconversion (de novo appearance of IgG antibodies) during pregnancy or by evidence of specific IgM antibodies in the initial serum specimen. Women who had IgG antibodies and no specific IgM antibodies at enrolment in the study were diagnosed as having recurrent (nonprimary) infection if they had a fourfold or greater rise in antibody titre during pregnancy or if the virus was isolated from clinical specimens collected during pregnancy, or both.
Amniocentesis
Methods Patient selection Sixteen pregnant women (13 with CMV infection) agreed to participate in the study. Twelve of the women with CMV infection were recruited from a population of 35 infected women identified during a prospective study in Montreal designed to determine the frequency of asymptomatic CMV infection in pregnant women.2' The study involved women receiving their prenatal care at four area medical centres: Centre hospitalier Fleury, H6pital Charles Le Moyne, H6pital Maisonneuve-Rosemont and H6pital Sainte-Justine. 650
CAN MED ASSOC J 1991; 145 (6)
Standard techniques were used to collect 5 to 10 mL of amniotic fluid for virus isolation at the amniocentesis units of the participating hospitals.
Identification ofinfection in the offspring Congenital infection was established through isolation of the virus from pharyngeal mucus and urine specimens collected during the first 3 days of life and, if the pregnancy was terminated, through isolation of the virus from fetal tissue. Clinical data were recorded for all the newborn babies. Children born of women whose amniotic fluid was found to be infected were followed up at regular intervals LE 15 SEPTEMBRE 1991
w-
until 2 to 3 years of age. At each visit urine, pharyngeal mucus and serum specimens were examined for evidence of infection. All specimens for virus isolation and serologic studies were tested at the Virology Research Centre, Institut Armand-Frappier.
Virus isolation
glutination-inhibition method.25 Serologic testing involved the simultaneous titration of all serum samples from each patient. A sucrose density-gradient technique was used to isolate the CMV-specific and the rubella-specific IgM antibodies.26 The CMV antibodies were then detected by means of indirect immunofluorescence assay27 and the rubella antibodies by means of a hemagglutination-inhibition technique.25
The amniotic fluid, pharyngeal mucus and urine specimens were kept at + 4°C and were inoculated Electron microscopy within 24 hours into appropriate,cell cultures. CMV Variations of the agar diffusion method28 and an was isolated in monolayer culture with human embryonic lung fibroblasts and identified by its patho- Airfuge method29 were used to prepare specimens for gnomonic cytopathic effect and its failure to repli- electron microscopy. Grids were examined for the cate in nonhuman or epithelial cells.22 The interfer- presence of virus particles at a magnification of ence method with echovirus type 11 was used for 20 000 to 40 000 using a Philips EM 300 electron isolation and identification of rubella virus after the microscope (Philips Electron Optics, Eindhoven, amniotic fluid was inoculated into three different Holland). At least 15 grid squares were examined for types of cell culture: primary African green monkey 15 minutes before the results were considered to be kidney (AGMK) cells, rabbit kidney cells of the negative. RK1 3 line and Vero continuous-line cells.23 In addition, all cultures positive for either virus were Results routinely examined by means of electron microscopy for the presence of particles of the appropriate virus CMV infection group.
Serologic studies Serum specimens were tested for CMV and rubella antibodies by means of a standard complement fixation technique24 and for rubella antibodies through the use of a previously described hemag-
CMV was isolated from the amniotic fluid of 3 of the 13 women who were infected with the virus (Table 1). These 3 women had primary CMV infection, whereas the other 10 had recurrent CMV infection. The first of the women with infected amniotic fluid (Table 1, case 9) had an uncomplicated preg-
Table 1: Results of serologic studies and culture of cytomegalovirus (CMV) from the amniotic fluid of CMV-infected women and from pharyngeal mucus and urine samples of their newborn infants
Case
Trimester during which Age, infection
no.
yr
1 2 3
24 31 28 24 25 21 21 32 26 24 27 21 19
4 5
6 7 8
9 10 11 12 13
Virus isolation
Initial antibody
detected 1 1 1 1 1 1 1 2 2 3 3
3 3
titre*
1:32 1:16 1:32 1:64 1:64 1:32 1:32 1:16
