Archives of Virology
Archives of Virology 53, 177--184 (1977)
© by Springer-Verlag 1977
IgM Antibodies to Cytomegalovirus Durin 9 Pregnancy By H. SCHMITZ, D. KA~II"A, H. W. DO~RR, T. LUTHARDT, H. G. HILLEMAI~ZgS,and A. WORTELE Zentrum f/ir Hygiene and Universit/its-Frauenklinik, Freiburg, Federal Republic of Germany With 2 Figures Accepted October 25, 1976 Sunnnary IgM antibodies to cytomegalovirus (CMV) could be detected in about 7 per cent of 629 pregnant women, whereas in 225 nonpregnant control women of similar age distribution only 2.6 per cent showed CMV IgM antibodies. Intrauterine CMV infections were almost exclusively detected among the CMV IgM positive gravides. The high incidence of CMV IgM antibodies in pregnant women can be possibly explained b y an increased rate of CMV reactivations during pregnancy. W~e were able to show that during CMV reactivation an intrauterine infection might occur. Introduction In most cases of congenital CMV infection, data on the preceeding virus multiplication in pregnant mothers do not exist. As a rule, virus multiplication in pregnant women is subclinical. To obtain more information on CMV intrauterine infections numerous prospective studies have been performed (2, 3, 7, 14). The investigators looked for a significant increase in titer in the complement-fixation test and/o r virus excretion in urine or cervical secretions. From these studies we have learned ~hat primary infection (or seroconversion) during pregnancy m a y in some cases result in congenital infection. The high frequency of CMV infections during pregnancy cannot completely be explained b y primary infections. A high rate of CMV infections is probably due to reactivations or even reinfec$ions. However, not much informat, ion is available about the number of reactivations or reinfections and their role in pregnancy. The introduction of virus-specific IgM determination in our routine diagnostics (10) has clearly shown t h a t CMV infections and especially reactivations or reinfections can be identified more often: 1. I n adults p r i m a r y infections regtflarly lead to the production of virusspecific IgM antibodies.
178
I-I. SCHMITZ et al. :
2. I n seropositive persons newly synthesized CMV IgM antibodies with or without a rise in the complement-fixing antibody titer indicate a secondary or anamnestic response. During our routine work CMV IgM antibodies h~d been found in an unexpectedly high frequency in serum specimens of pregnant women. We therefore started a prospective study on CMV Igl~i antibodies during pregnancy and we also extended our s~udy to the babies. Materials and Methods Sera
874 sera of 629 p r e g n a n t w h i t e w o m e n were collected during their routine a t t e n d e n c e at t h e U n i v e r s i t y W o m a n ' s H o s p i t a l Freiburg. 135 umbilical cord blood sera could be o b t a i n e d f r o m the babies respectively. 225 sera were t a k e n f r o m h e a l t h y n o n - p r e g n a n t w o m e n of similar age distribution (18---37 years) as in the p r e g n a n t group. All sera were stored at. - - 2 0 ° C. Virus Isolation I n m o s t babies w i t h CMV IgM antibodies in the umbilical cord blood urine samples were collected and virus isolations were carried out on h u m a n fibroblast cells as has been published (10). Immuno]luorescence Tests Tile techniques of t h e CMV I g M and I g G a n t i b o d y tests h a v e been described in detail elsewhere (8). F o r t h e d e t e c t i o n of CMV I g M antibodies the double indirect m e t h o d was e m p l o y e d using a r a b b i t globulin fraction against h u m a n ~-chains (dilution 1 : 60) and a fluorescein labelled I g G fraction against r a b b i t I g G of swine origin (both f r o m D a k o p a t t s , Copenhagen). As c o m p a r e d to t h e indirect t e c h n i q u e using fluorescein labelled anti ~z-chain globulin, t h e double indirect m e t h o d is m u c h cheaper' because a considerably higher dilution of t h e unlabelled anti ~-chain globulin can be applied. I t should be stressed t h a t cell smears instead of coverslip cultures cannot be used. I n smears the n u c l e a r inclusions are n o t well differentiated f r o m o t h e r fluorescing m a t e r i a l and the p e n e t r a t i o n of t h e I g M antibodies t h r o u g h t h e t h i c k e n e d cell walls of t h e trypsinized cells is inhibited in b o t h directions. T h e p e n e t r a t i o n of the large IgM molecules can be f u r t h e r i m p r o v e d b y p u t t i n g t h e coverslip cultures prior to acetone fixation (15 minutes, - - 2 0 ° C) in a solution of 0.4 per cent KC1 (in a dest., 3 minutes, 20 ° C), which leads to a considerable swelling of the cells.
Complement Fixation Test The test was carried out in a m i c r o t i t e r s y s t e m using CMV antigen of Behring %Verke, Marburg, G e r m a n y (8, 10). Speei]icity o] Low CM V IgJVl Antibody Titers Low IgM antibody titers were often found in the sera of tile pregnant women. It had to be proven, therefore, that even low titers could be specifically detected. Especially in low dilutions of human sera with high quantities of total IgM a nonspecific deposition of the IgM molecules at the nuclear inclusions of the infected cells might occur during imraunofluoreseenee testing. However, non-specifically deposited IgM would not fix complement. Therefore, in 31 sera with CMV IgM antibody titers of 1:16--1:64 the IgM fractions were isolated by sucrose (gradient centrifugation (I i) and then tested in an anti-complement immunofluorescence (ACId') test, a technique, which has turned out to be of value for the detection of antibodies to Epstein-Barr virus nuclear antigen (6). The technical details of this method are described elsewhere (8, 12).
IgM to CMV During Pregnancy
179
Results First of all it had to be proven that low serum dilutions would not produce falsely positive results in the double indirect immunofluorescence test. Therefore, we looked for the presence of CMV IgM antibodies in 31 serum specimens with low (1:16--1:64) IgM antibody titers in the double indirect immunofluorescence method by an additional technique. Table 1 shows that, with only one exception, in all sera with CMV IgM antibody titers of 1 : 32 and 1 : 64 in the double indirect test, CMV IgM antibodies could also be detected by the more reliable but time consuming testing of the isolated IgM fractions with the ACIF method. Titers of 1 : ~32 are specifically detected with the routinely used double indirect immunofluorescence method. In Table 2 the IgM response in the first 629 sera of all pregnant women studied and in the 225 sera of the nonpregnant control women are listed. Specific IgM antibodies (titer 1:/>32) could be detected in 6--7 per cent of the pregnant women in each trimester. The high percentage of the IglVf positives during pregnancy differs significantly (t-test : p ~
Archives of Virology 53, 177--184 (1977)
© by Springer-Verlag 1977
IgM Antibodies to Cytomegalovirus Durin 9 Pregnancy By H. SCHMITZ, D. KA~II"A, H. W. DO~RR, T. LUTHARDT, H. G. HILLEMAI~ZgS,and A. WORTELE Zentrum f/ir Hygiene and Universit/its-Frauenklinik, Freiburg, Federal Republic of Germany With 2 Figures Accepted October 25, 1976 Sunnnary IgM antibodies to cytomegalovirus (CMV) could be detected in about 7 per cent of 629 pregnant women, whereas in 225 nonpregnant control women of similar age distribution only 2.6 per cent showed CMV IgM antibodies. Intrauterine CMV infections were almost exclusively detected among the CMV IgM positive gravides. The high incidence of CMV IgM antibodies in pregnant women can be possibly explained b y an increased rate of CMV reactivations during pregnancy. W~e were able to show that during CMV reactivation an intrauterine infection might occur. Introduction In most cases of congenital CMV infection, data on the preceeding virus multiplication in pregnant mothers do not exist. As a rule, virus multiplication in pregnant women is subclinical. To obtain more information on CMV intrauterine infections numerous prospective studies have been performed (2, 3, 7, 14). The investigators looked for a significant increase in titer in the complement-fixation test and/o r virus excretion in urine or cervical secretions. From these studies we have learned ~hat primary infection (or seroconversion) during pregnancy m a y in some cases result in congenital infection. The high frequency of CMV infections during pregnancy cannot completely be explained b y primary infections. A high rate of CMV infections is probably due to reactivations or even reinfec$ions. However, not much informat, ion is available about the number of reactivations or reinfections and their role in pregnancy. The introduction of virus-specific IgM determination in our routine diagnostics (10) has clearly shown t h a t CMV infections and especially reactivations or reinfections can be identified more often: 1. I n adults p r i m a r y infections regtflarly lead to the production of virusspecific IgM antibodies.
178
I-I. SCHMITZ et al. :
2. I n seropositive persons newly synthesized CMV IgM antibodies with or without a rise in the complement-fixing antibody titer indicate a secondary or anamnestic response. During our routine work CMV IgM antibodies h~d been found in an unexpectedly high frequency in serum specimens of pregnant women. We therefore started a prospective study on CMV Igl~i antibodies during pregnancy and we also extended our s~udy to the babies. Materials and Methods Sera
874 sera of 629 p r e g n a n t w h i t e w o m e n were collected during their routine a t t e n d e n c e at t h e U n i v e r s i t y W o m a n ' s H o s p i t a l Freiburg. 135 umbilical cord blood sera could be o b t a i n e d f r o m the babies respectively. 225 sera were t a k e n f r o m h e a l t h y n o n - p r e g n a n t w o m e n of similar age distribution (18---37 years) as in the p r e g n a n t group. All sera were stored at. - - 2 0 ° C. Virus Isolation I n m o s t babies w i t h CMV IgM antibodies in the umbilical cord blood urine samples were collected and virus isolations were carried out on h u m a n fibroblast cells as has been published (10). Immuno]luorescence Tests Tile techniques of t h e CMV I g M and I g G a n t i b o d y tests h a v e been described in detail elsewhere (8). F o r t h e d e t e c t i o n of CMV I g M antibodies the double indirect m e t h o d was e m p l o y e d using a r a b b i t globulin fraction against h u m a n ~-chains (dilution 1 : 60) and a fluorescein labelled I g G fraction against r a b b i t I g G of swine origin (both f r o m D a k o p a t t s , Copenhagen). As c o m p a r e d to t h e indirect t e c h n i q u e using fluorescein labelled anti ~z-chain globulin, t h e double indirect m e t h o d is m u c h cheaper' because a considerably higher dilution of t h e unlabelled anti ~-chain globulin can be applied. I t should be stressed t h a t cell smears instead of coverslip cultures cannot be used. I n smears the n u c l e a r inclusions are n o t well differentiated f r o m o t h e r fluorescing m a t e r i a l and the p e n e t r a t i o n of t h e I g M antibodies t h r o u g h t h e t h i c k e n e d cell walls of t h e trypsinized cells is inhibited in b o t h directions. T h e p e n e t r a t i o n of the large IgM molecules can be f u r t h e r i m p r o v e d b y p u t t i n g t h e coverslip cultures prior to acetone fixation (15 minutes, - - 2 0 ° C) in a solution of 0.4 per cent KC1 (in a dest., 3 minutes, 20 ° C), which leads to a considerable swelling of the cells.
Complement Fixation Test The test was carried out in a m i c r o t i t e r s y s t e m using CMV antigen of Behring %Verke, Marburg, G e r m a n y (8, 10). Speei]icity o] Low CM V IgJVl Antibody Titers Low IgM antibody titers were often found in the sera of tile pregnant women. It had to be proven, therefore, that even low titers could be specifically detected. Especially in low dilutions of human sera with high quantities of total IgM a nonspecific deposition of the IgM molecules at the nuclear inclusions of the infected cells might occur during imraunofluoreseenee testing. However, non-specifically deposited IgM would not fix complement. Therefore, in 31 sera with CMV IgM antibody titers of 1:16--1:64 the IgM fractions were isolated by sucrose (gradient centrifugation (I i) and then tested in an anti-complement immunofluorescence (ACId') test, a technique, which has turned out to be of value for the detection of antibodies to Epstein-Barr virus nuclear antigen (6). The technical details of this method are described elsewhere (8, 12).
IgM to CMV During Pregnancy
179
Results First of all it had to be proven that low serum dilutions would not produce falsely positive results in the double indirect immunofluorescence test. Therefore, we looked for the presence of CMV IgM antibodies in 31 serum specimens with low (1:16--1:64) IgM antibody titers in the double indirect immunofluorescence method by an additional technique. Table 1 shows that, with only one exception, in all sera with CMV IgM antibody titers of 1 : 32 and 1 : 64 in the double indirect test, CMV IgM antibodies could also be detected by the more reliable but time consuming testing of the isolated IgM fractions with the ACIF method. Titers of 1 : ~32 are specifically detected with the routinely used double indirect immunofluorescence method. In Table 2 the IgM response in the first 629 sera of all pregnant women studied and in the 225 sera of the nonpregnant control women are listed. Specific IgM antibodies (titer 1:/>32) could be detected in 6--7 per cent of the pregnant women in each trimester. The high percentage of the IglVf positives during pregnancy differs significantly (t-test : p ~
