THE JOURNAL OF INFECTIOUS DISEASES • VOL. 132, NO.5· © 1975 by the University of Chicago. All rights reserved.
NOVEMBER 1975
Comparative Serial Virologic and Serologic Studies of Symptomatic and Subclinical Congenitally and Natally Acquired Cytomegalovirus Infections Sergio Stagno, David W. Reynolds, Alex Tsiantos, * David A. Fuccillo, Walter Long, and Charles A. Alford
From the Department of Pediatrics and the Clinical Research Center, School of Medicine. University of Alabama in Birmingham, Birmingham, Alabama; and the Section of Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland
Although clinical and epidemiological knowledge of cytomegalovirus (CMV) infection is
rapidly expanding, host-parasite relations remain poorly understood [I]. Definition of the natural history is particularly important in perinatal populations because of the high incidence and the potential morbidity associated with this type of infection. Congenital involvement ranges between 0.5% and 2% of all live births [2-5]. Natal infection (i.e., mother-tochild transmission at delivery from a CMVinfected genital tract) accounts for an additional 5%, at least in our own low socioeconomic population [6]. Congenital infection only occasionally causes appreciable acute morbidity in the neonate, but sequelae, including decreased mentation and auditory impairment, often supervene even among patients with subclinical presentation at birth [7, 8J. The possible deleterious effects of the natally acquired infections have yet to be defined. Previous natural history studies have been
This paper is dedicated to the memory of Dr. Alexander Tsiantos. Received for publication May 7, 1975, and in revised form July 24, 1975. This work was supported in part by research grant no. H 0-01687 and training grant no. H 0-00413 from the National Institute of Child Health and Human Development; by Public Health Service grant no. MOl RR00032 from the General Clinical Research Centers; by research grant no. CA-13l48 from the National Cancer Institute; by a research grant from the National Foundation-March of Dimes; and by the Robert E. Meyer Foundation. We thank Mr. Rolfe Reynolds and Mr. Richard J. Smith for technical assistance and Ms. Betty Smith and Ms. Connie Curtis for assistance in the preparation of the manuscript. Piease address requests for reprints to Dr. Sergio Stagno, University of Alabama in Birmingham, Medical Center, C.D.L.D. Building, Room 609, University Station, Birmingham, Alabama 35294. * Deceased.
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Infants with congenitally (38) and natally (17) acquired cytomegalovirus infection were prospectively studied by means of virologic and multiple serologic assays, These infections were characterized by chronic viral excretion (measured in years). The quantity of virus excreted in the urine during early infancy was significantly greater in infants who acquired infection in utero, particularly among those born with overt disease; thereafter, all three groups (congenital symptomatic, congenital asymptomatic, and natal) excreted similar amounts of virus, The patterns of antibody responses, particularly the fluorescent antibody response to the early antigen and the complement-fixing antibody response, further indicated that congenitally infected infants (especially symptomatic ones) bear a greater antigenic burden than do natally infected infants. From a diagnostic standpoint, the test for fluorescent antibody to the late antigen was the most sensitive assay, whereas the test for complement-fixing antibody proved to be the least useful. The indirect hemagglutination assay, although performed only in infants with natal infection, was only slightly less sensitive than the fluorescent antibody procedure; by the former technique, diagnostic rises were detected in all but one infant after the onset of viruria.
Virology and Serology of CMV Infection
Materials and Methods
Patient population and specimens. The population studied included 38 infants with congenital infection and 17 infants with natal infection. The former group (mean age, 35 months; range, four to 84 months) was identified by means of an umbilical cord IgM screening method, and the judgment was confirmed by the presence of viruria during the first week of life [7]. Only eight of the 38 infants presented with CMV -related symptomatology at birth. Infants with natal C MV infection (mean age, 15 months; range, four to 30 months) were born to mothers proven to have genital CMV infection near the time of delivery. Urine specimens collected from infants at an average of two days of age and again at 15 days failed to yield CMV; however, the majority of the infants became viruric between four and eight weeks of age [6]. All have remained asymptomatic. Infants from both groups were seen at one, two, three, four, five, six, nine, and 12 months of age and twice a year thereafter. At each clinic visit urine and blood specimens were collected for viral quantitation and serologic analysis, respectively.
Controls. Twenty infants proven to be virologically uninfected were followed longitudinally as controls from birth to 15months of age. Similar virologic and serologic assessments were made at trimonthly intervals. Virology and serology. Urine specimens were processed and examined; two tubes of monolayer cultures of human skin fibroblasts per specimen were used [6]. Urine titrations were made with three tubes of cell culture per to-fold dilution, and results were expressed as TCIDsol 0.2 ml of urine inoculum. CMV was identified by its characteristic CPE in hematoxylin and eosinstained or viable preparations and by its failure to replicate in nonhuman and human epithelial cells. Serology. The AD-169 strain of CMV was employed as the source of antigen for all serologic assays. The CF test was performed according to the micromethod of Melnick [9]; two full units of complement and four CF units of antigen, as determined with a known positive serum, were used. Antigen was prepared in human fibroblast monolayers according to the method of Benyesh- Melnick [10]. Briefly, when CPE reached 80%-90%, the cell monolayers were trypsinized, suspended in veronal buffer, and then disrupted by sonication for 10 sec at 25 W (Sonifier Cell Disruptor, model W185, Heat Systems- Ultrasonics, Plainview, N. Y.). Supernatant fluid containing the antigen was clarified by centrifugation at 280 g for 15 min; 0.5-ml aliquots were stored at -70 C until used. Only two batches of antigen were used. Sheep red blood cells, hemolysin, and guinea pig complement were obtained commercially (Colorado Serum Co., Denver, Colo.). The IRA test was performed as previously described [11]. Only the sera of patients with natal infection were studied by means of this assay. The indirect F A assay was used for measurement of antibodies produced against late and early CMV-induced antigens. Target cells were prepared according to the method of The et al. [12]. Briefly, confluent monolayers of human fibroblasts grown in 75-cm 2 plastic flasks (Falcon Plastics, Oxnard, Calif.) were exposed to 10 ml of supernatant CMV (multiplicity of input, approximately 1) of the AD-169 strain for
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on August 28, 2015
confined to patients with congenital infections and for the most part have correlated their clinical outcome with duration of viral excretion and humoral immune response, as measured by the CF test and occasionally by neutralization and the IgM fluorescent antibody (F A) assays [5, 8]. The present longitudinal study comprises patients with natal and both symptomatic and subclinical congenital infections. To elucidate further some characteristics of the virus-host interaction, we have correlated not only the duration of viral excretion but also the quantity of virus excreted over time by these patients with their humoral immune response. The latter was longitudinally assessed by CF, FA (using early and late antigens), and indirect hemagglutination (IRA) assays. These newer techniques were employed in the hope of better defining the host response to the infection and because of their practical value with respect to differentiation of subclinical congenital infection from that acquired at delivery.
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1 hr at 37 C; 25 ml of maintenance medium (199 Earle's) was added. Infected monolayers for the preparation of late antigen were harvested by trypsinization (as described below) when CPE reached 80%-90%, usually on the fourth day.
Congenitally infected patients. (I) Excretion of virus. From the 38 patients with congenital CMV infection, a total of 196 urine samples were examined (mean, 5.1 samples per patient). In both symptomatic and subclinically infected infants, viral excretion was universal at birth. In the latter group, virus shedding decreased slightly during the first four years of life; in fact, 15 of 17, 20 of 22, 16 of 18, and nine of 12 infants remained viruric when tested at one, two, three, and four years of age, respectively. After four years, only two of the six children examined continued to excrete virus intermittently (for 54 and 72 months, respectively). All of the four symptomatic cases followed for longer than 12 months have continued to shed virus in the urine (age range, 30-72 months). One hundred twenty-one urine samples were available for viral titration (mean, 3.2 samples per patient). As is shown in figure I, viral output in infants with subclinical congenital infection ranged from 4.3 to 4.5 Iogs during the first three months of life. Later output dropped progressively and reached a stable level when infants were nine months of age. Although observations are limited beyond 30 months, the quantity of virus excreted appears to be waning. Among patients with symptomatic congenital infection, excretion of CMV was significantly higher at birth and during the early months of life (P
NOVEMBER 1975
Comparative Serial Virologic and Serologic Studies of Symptomatic and Subclinical Congenitally and Natally Acquired Cytomegalovirus Infections Sergio Stagno, David W. Reynolds, Alex Tsiantos, * David A. Fuccillo, Walter Long, and Charles A. Alford
From the Department of Pediatrics and the Clinical Research Center, School of Medicine. University of Alabama in Birmingham, Birmingham, Alabama; and the Section of Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland
Although clinical and epidemiological knowledge of cytomegalovirus (CMV) infection is
rapidly expanding, host-parasite relations remain poorly understood [I]. Definition of the natural history is particularly important in perinatal populations because of the high incidence and the potential morbidity associated with this type of infection. Congenital involvement ranges between 0.5% and 2% of all live births [2-5]. Natal infection (i.e., mother-tochild transmission at delivery from a CMVinfected genital tract) accounts for an additional 5%, at least in our own low socioeconomic population [6]. Congenital infection only occasionally causes appreciable acute morbidity in the neonate, but sequelae, including decreased mentation and auditory impairment, often supervene even among patients with subclinical presentation at birth [7, 8J. The possible deleterious effects of the natally acquired infections have yet to be defined. Previous natural history studies have been
This paper is dedicated to the memory of Dr. Alexander Tsiantos. Received for publication May 7, 1975, and in revised form July 24, 1975. This work was supported in part by research grant no. H 0-01687 and training grant no. H 0-00413 from the National Institute of Child Health and Human Development; by Public Health Service grant no. MOl RR00032 from the General Clinical Research Centers; by research grant no. CA-13l48 from the National Cancer Institute; by a research grant from the National Foundation-March of Dimes; and by the Robert E. Meyer Foundation. We thank Mr. Rolfe Reynolds and Mr. Richard J. Smith for technical assistance and Ms. Betty Smith and Ms. Connie Curtis for assistance in the preparation of the manuscript. Piease address requests for reprints to Dr. Sergio Stagno, University of Alabama in Birmingham, Medical Center, C.D.L.D. Building, Room 609, University Station, Birmingham, Alabama 35294. * Deceased.
568
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on August 28, 2015
Infants with congenitally (38) and natally (17) acquired cytomegalovirus infection were prospectively studied by means of virologic and multiple serologic assays, These infections were characterized by chronic viral excretion (measured in years). The quantity of virus excreted in the urine during early infancy was significantly greater in infants who acquired infection in utero, particularly among those born with overt disease; thereafter, all three groups (congenital symptomatic, congenital asymptomatic, and natal) excreted similar amounts of virus, The patterns of antibody responses, particularly the fluorescent antibody response to the early antigen and the complement-fixing antibody response, further indicated that congenitally infected infants (especially symptomatic ones) bear a greater antigenic burden than do natally infected infants. From a diagnostic standpoint, the test for fluorescent antibody to the late antigen was the most sensitive assay, whereas the test for complement-fixing antibody proved to be the least useful. The indirect hemagglutination assay, although performed only in infants with natal infection, was only slightly less sensitive than the fluorescent antibody procedure; by the former technique, diagnostic rises were detected in all but one infant after the onset of viruria.
Virology and Serology of CMV Infection
Materials and Methods
Patient population and specimens. The population studied included 38 infants with congenital infection and 17 infants with natal infection. The former group (mean age, 35 months; range, four to 84 months) was identified by means of an umbilical cord IgM screening method, and the judgment was confirmed by the presence of viruria during the first week of life [7]. Only eight of the 38 infants presented with CMV -related symptomatology at birth. Infants with natal C MV infection (mean age, 15 months; range, four to 30 months) were born to mothers proven to have genital CMV infection near the time of delivery. Urine specimens collected from infants at an average of two days of age and again at 15 days failed to yield CMV; however, the majority of the infants became viruric between four and eight weeks of age [6]. All have remained asymptomatic. Infants from both groups were seen at one, two, three, four, five, six, nine, and 12 months of age and twice a year thereafter. At each clinic visit urine and blood specimens were collected for viral quantitation and serologic analysis, respectively.
Controls. Twenty infants proven to be virologically uninfected were followed longitudinally as controls from birth to 15months of age. Similar virologic and serologic assessments were made at trimonthly intervals. Virology and serology. Urine specimens were processed and examined; two tubes of monolayer cultures of human skin fibroblasts per specimen were used [6]. Urine titrations were made with three tubes of cell culture per to-fold dilution, and results were expressed as TCIDsol 0.2 ml of urine inoculum. CMV was identified by its characteristic CPE in hematoxylin and eosinstained or viable preparations and by its failure to replicate in nonhuman and human epithelial cells. Serology. The AD-169 strain of CMV was employed as the source of antigen for all serologic assays. The CF test was performed according to the micromethod of Melnick [9]; two full units of complement and four CF units of antigen, as determined with a known positive serum, were used. Antigen was prepared in human fibroblast monolayers according to the method of Benyesh- Melnick [10]. Briefly, when CPE reached 80%-90%, the cell monolayers were trypsinized, suspended in veronal buffer, and then disrupted by sonication for 10 sec at 25 W (Sonifier Cell Disruptor, model W185, Heat Systems- Ultrasonics, Plainview, N. Y.). Supernatant fluid containing the antigen was clarified by centrifugation at 280 g for 15 min; 0.5-ml aliquots were stored at -70 C until used. Only two batches of antigen were used. Sheep red blood cells, hemolysin, and guinea pig complement were obtained commercially (Colorado Serum Co., Denver, Colo.). The IRA test was performed as previously described [11]. Only the sera of patients with natal infection were studied by means of this assay. The indirect F A assay was used for measurement of antibodies produced against late and early CMV-induced antigens. Target cells were prepared according to the method of The et al. [12]. Briefly, confluent monolayers of human fibroblasts grown in 75-cm 2 plastic flasks (Falcon Plastics, Oxnard, Calif.) were exposed to 10 ml of supernatant CMV (multiplicity of input, approximately 1) of the AD-169 strain for
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on August 28, 2015
confined to patients with congenital infections and for the most part have correlated their clinical outcome with duration of viral excretion and humoral immune response, as measured by the CF test and occasionally by neutralization and the IgM fluorescent antibody (F A) assays [5, 8]. The present longitudinal study comprises patients with natal and both symptomatic and subclinical congenital infections. To elucidate further some characteristics of the virus-host interaction, we have correlated not only the duration of viral excretion but also the quantity of virus excreted over time by these patients with their humoral immune response. The latter was longitudinally assessed by CF, FA (using early and late antigens), and indirect hemagglutination (IRA) assays. These newer techniques were employed in the hope of better defining the host response to the infection and because of their practical value with respect to differentiation of subclinical congenital infection from that acquired at delivery.
569
Stagno et al.
570
1 hr at 37 C; 25 ml of maintenance medium (199 Earle's) was added. Infected monolayers for the preparation of late antigen were harvested by trypsinization (as described below) when CPE reached 80%-90%, usually on the fourth day.
Congenitally infected patients. (I) Excretion of virus. From the 38 patients with congenital CMV infection, a total of 196 urine samples were examined (mean, 5.1 samples per patient). In both symptomatic and subclinically infected infants, viral excretion was universal at birth. In the latter group, virus shedding decreased slightly during the first four years of life; in fact, 15 of 17, 20 of 22, 16 of 18, and nine of 12 infants remained viruric when tested at one, two, three, and four years of age, respectively. After four years, only two of the six children examined continued to excrete virus intermittently (for 54 and 72 months, respectively). All of the four symptomatic cases followed for longer than 12 months have continued to shed virus in the urine (age range, 30-72 months). One hundred twenty-one urine samples were available for viral titration (mean, 3.2 samples per patient). As is shown in figure I, viral output in infants with subclinical congenital infection ranged from 4.3 to 4.5 Iogs during the first three months of life. Later output dropped progressively and reached a stable level when infants were nine months of age. Although observations are limited beyond 30 months, the quantity of virus excreted appears to be waning. Among patients with symptomatic congenital infection, excretion of CMV was significantly higher at birth and during the early months of life (P
