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Respiratory tract excretion of cytomegalovirus in Thai children
TRO P ICA L
P E D I A T R I C S DerrickB. Jelliffe,
Editor
Respiratory tract excretion of cytomegalovirus in Thai children Cytomegalovirus has been found to be commonly present in the respiratory tract secretions of Thai children. Eleven per cent of children with pertussis syndrome, 18.1 per cent of 342 children with respiratory tract infections, and 15.1 per cent of well babies were positive [or cytomegalovirus. Virus was most commonly present in children less than one year of age, the incidence falling progressively with age so that it was unusual to recover cytomegalovirus #ore children over the age of 4 years. Most patients tested possessed serum neutralizing and immunoglobulin M antibody at the time virus was being excreted, but few had complement-fixing antibody.
Lloyd C. Olson, M.D., R a m p e y a Ketusinha, M.D.,
Pethai Mansuwan, M.D., and Rapin Snitbhan, M.D. BANGKOK,
THAILAND
A L W~ O U a H T H E R E is considerable evidence to indicate that cytomegalovirus infection in m a n is a common occurrence, little is known as to how and when individuals of a population become infected. In particular, the role of excretion of cytomegalovirus from the respiratory tract as a means of transmission remains unknown. Salivary gland infection has been demonstrated repeatedly, 1 although the rarity with which this agent has been recovered during virologic studies of respiratory tract flora might suggest that inFrom the Department of Virology, S E A T O Medical Research Laboratory; Children's Hospital; and Faculty of Public Health, Mahidol University. Rtpr~nt address: L. C. Olson, USA Medical Component SEATO, APO San Francisco, Calif. 96346.
fectious virus does not gain access to the environment from this site. 2 During a longitudinal study of the respiratory viruses of children living in Bangkok, an unexpected number yielded cytomegalovirus from the respiratory tract. This report details isolation rates of this virus from various groups of children living in this tropical locale. M A T E R I A L AND M E T H O D S Patient population. Children from whom nasopharyngeal swabs were obtained were part of 3 groups at the ChiIdren's Hospital, Bangkok, being studied for respiratory viruses. Those with upper respiratory infections were selected for study by a single VoI. 77, No. 3, pp. 499-504
500
Olson et al.
physician in the outpatient department if they were under the age of 12 years and had been ill less than 48 hours with fever and respiratory tract symptoms. Patients who had clinical symptoms consistent with the diagnosis of whooping cough of less than 3 weeks' duration were studied as a separate group. Infants in the newborn nursery were arbitrarily selected for study. Well children were selected from the well baby clinic if, by history and physical examination, they were free of symptoms. Virologic procedures. Nasopharyngeal swabs were obtained and immediately placed in 2.0 ml. of Hanks balanced salt solution containing 0.4 per cent bovine plasma albumin. Urine was collected from newborn infants and centrifuged lightly to remove debris. All specimens were treated with antibiotics (penicillin-streptomycin-neomycin) and 0.1 ml. was inoculated into each of 3 tube cultures of WI-38 cells (originally obtained from Dr. Leonard Hayflick, Wistar Institute). After absorption overnight at 37 ~ C., media were decanted and replaced with medium L-15 containing 2 per cent heatinactivated fetal bovine serum and antibiotics (penicillin-streptomycin-amphotericin B). Cultures were thereafter incubated at 33 ~ C., observed microscopically for evidence of cytopathic effects twice weekly, and fed with fresh maintenance media at intervals of 1 to 2 weeks. Cultures were considered negative only if cell monolayers were maintained in satisfactory condition for at least 6 weeks without any evidence of focal cytopathic effects. Virus identification. Cytomegalovirus was presumptively identified on the basis of cytopathic effect characteristics, as described by others, a and usually appeared in the second to fourth week after inoculation. Cytopathic effect was allowed to progress until it involved most of the cell sheet or until cells otherwise began to spontaneously degenerate, at which times cells were scraped into the media, disrupted by freezing and thawing rapidly 3 times, centrifuged, and the supernatant passed to fresh cell cultures. On passage the belated reappearance of char-
The Journal of Pediatrics September 1970
acteristic cytopathic effects was taken as confirmatory evidence of the presence of cytomegalovirus. Some procedures were admittedly not those conventionally employed for cytomegalovirus, but originally these were designed as a detection system for rhinoviruses. When it became obvious that cytomegalovirus was frequently present, a series of 78 specimens from the group of children with upper respiratory infections were inoculated separately into 2 groups of cell cultures and incubated at 33 and 37 ~ C., respectively. While cytopathic effects appeared more slowly at 33 ~ C., the same 16 specimens were ultimately positive in both groups. Thus, in order to maintain a uniform procedure, incubation at 33 ~ C. was continued. In no instance did a primary isolate of cytomegalovirus fail to pass after freeze-thaw. O n occasion, adenovirus cytopathic effect was initially mistaken for the cytopathic effect of cytomegalovirus. The much more rapid growth in WI-38 ceils and the ability to produce cytopathic effects in human heteroploid cell lines then made adenoviruses easily recognizable. Complement fixation tests. Complement fixation antibody was determined using, in separate tests, both an antigen prepared commercially from AD-169 strain (Microbiological Associates, Rockville, Md.) and from a locally isolated strain using a method similar to that of Lang and Noren. 4 Tests were carried out in disposable microtiter plates using 2 to 4 units of antigen with overnight fixation. Neutralizing antibody tests. Screening tests for serum neutralizing antibody on heat-inactivated serum (56 ~ C., 30 minutes) were carried out at a serum dilution of 1:4 by mixing with approximately 100 tissue culture 50 per cent doses (TCIDs0) of one of the strains isolated in this study (strain No. 154). After reacting at room temperature for one hour, the mixture was inoculated into replicate tube cultures of WI-38 cells. Tests were read weekly for 4 weeks. Consistently obvious differences in the amount of cytopathic effect in the test specimens in
Volume 77 Number 3
Excretion o[ cytornegalovirus
Table I. Recovery of cytomegalovirus by age groups in patients with upper respiratory infections Age
< l yr. 1
2 3 >4
Totals
No. cultured
No. positive
81 86 51 37 87 342
32 17 6 5 2 62
Table ]I. Recovery of cytomegalovirus by age groups in patients with pertussis syndrome No. cultured
No. positive
%
Age
39.5
<1 yr.
12
4
19.8
1
19
3
11.8 13.5 2.3 18.1
2 3 >4 Total
14 12 43 100
2 I 1 11
contrast to that in the control specimens was considered as evidence of antibody. Indirect immunofluorescent antibody tests. In the tests for fluorescent antibody a local virus strain (No. 154) was employed; the method followed that of Hanshaw and associates 5 with the following modifications. Tests were carried out using separately goat antihuman gamma globulin and goat antihuman IgM (Hyland Laboratories, Los Angeles, Calif.) after conjugation with fluorescein isothiocyanate. 6 Sera to be tested were diluted 1:10 in phosphate-buffered saline p H 7.2 and adsorbed for 2 hours at room temperature on tube monolayers of WI-38 cells previously washed 3 times with phosphate buffered saline. Fluid was then aspirated and tested without further treatment. This procedure was found to remove effectively nonspecific immunofluorescence from most sera. Goat sera were adsorbed with WI-38 cell suspensions. ~ RESULTS
In the group of children studied with upper respiratory tract infections, of the approximately 45,000 children seen with this diagnosis in the clinic of Children's Hospital, 342 were studied from January to December, 1968. From this group, cytomegalovirus was isolated from 62 patients, or 18.1 per cent of those studied. Forty of the patients were boys and 22 were girls. Virus was isolated most frequently from children less than one year of age, the per cent positive progres-
50 1
%
33.3 16.0 14.3 8.3 2.3 11.0
slvely dropping with increasing age so that it was unusual to recover virus after the age of 4 years (Table I). Similar recovery rates and age distribution of patients who were positive for cytomegalovirus were found during the period from July, 1968, to January, 1969, in 100 patients with a clinical diagnosis of whooping cough (Table II). In this group, 8 of the cytomegalovirus positive patients were girls and 3 were boys. Patients in the group with upper respiratory infections were also studied for the presence of other respiratory virusesY From the 62 patients excreting cytomegalovirus, another virus was isolated 17 times, while cultures of 91 of the 280 cytomeglovirusnegative patients were positive. With the exception of a rubella epidemic early in the year and the appearance of the 1968 A2 influenza variant in September, there were no unusual features of respiratory diseases throughout this year as compared to other years in the community. Virus was recovered from nasopharyngeal swabs from 10 of 66 well children under the age of one year studied during the period from August to November, 1968. This suggests a lower frequency of virus excretion in asymptomatic children, but the smaller size of this group and dissimilarity of age composition by month renders further analysis impossible (Table III). To rule out the unlikely possibility that virus recovered represented congenitally acquired infection, 187 urine samples were
502
Olson et al.
The Journal o] Pediatrics September t970
Table I l L Cytomegalovirus-positive patients, by month of age
Age (too.)
No. positive/No, cultured Upper respiratory infections group Well babies
1
2 3 4 5 6 7 8 9 10 11
1/I 1/2 4/7 9/13 6/17 2/11 7/17 2/13 32/81
0/6 1/12 4/11 1/10 2/10 I/2 1/5 O/5 O/4 0/I 10/66
collected from randomly selected newborn infants in July and October, 1968. Only 2 were positive for virus. Paired sera (acute phase and 2 weeks later) were collected from children in the upper respiratory infections and pertussis study groups. Thirty-four pairs from the upper respiratory infections group and 9 from the pertussis group, all from cytomegalovirus excretors, were tested (Table I V ) . Testing of the acute sera showed complement fixation antibody in sera of 4 of 9 pertussis patients, and 5 of tile 34 in the upper respiratory infections group. However, sera obtained 2 weeks later showed no increase in titer in sera from the latter group, while 2 of the pertussis patients had fourfold increases in complement fixation antibody, No differences in reactivity between the local strain and AD-169 were were found. Neutralizing antibody was present in most acute phase sera. Of the 38 sera demonstrating neutralizing antibody, 37 were confirmed by the demonstration of anticytomegalovirus globulin by immunofluorescence. Of these 37, anticytomegalovirus I g M was present in 26 of the sera at the dilution
Table IV. Antibody determinations in cytomegalovirus-positive patients
Determinations
Total tested Neutralizing antibody pos. FAT: globulin poe. FAT: IgM poe, Total CF poe, CF pos. IgM FAT pos.
Upper respiratory infections Pertussis group group
34 30 29 24
9 8 8 2
5 0
7 0
Total CF neg. 29 2 CF neg. IgM pos. 21 2 FAT = fluorescent antibody te~t. CF = complementfixing antibody.
tested. None of the sera which were complement fixation positive were also positive for IgM. Four acute phase sera were negative in all tests. All sera positive by neutralization tests were also positive by at least one other method; one serum was positive only for IgM. DISCUSSION Surveys for the presence of cytomegalovirus in the respiratory tracts of children in Bangkok have shown the virus to be commonly present, having been detected in 21.2 per cent of 378 children less than 4 years of age. Recovery rates were highest from infants less than one year of age: 40 per cent of those with upper respiratory infections, 33 per cent with a pertussis syndrome, and 15 per cent of well babies. T h e high frequency with which virus was recovered from young children in a single sample implies virus is present over a prolonged period of time, although serial follow-up will be necessary for confirmation. Although cytomegalovirus was recovered more frequently from children with upper respiratory infections, there is no evidence to suggest its presence was related to symptoms. In fact the isolation of other respiratory viruses
Volume 77 Number 3
from 30 per cent of the patients, irrespective of whether they were shedding cytomegalovirus or not, would argue against this. Other studies of viruses from the respiratory tract s, s have rarely or never encountered cytomegalovirus in children. It is doubtful if this is simply because the virus was not detected in other studies since, in the present study, many specimens were positive even within the usual observation period of 2 weeks. Another possibility explaining the high incidence might be that the current study coincided with an epidemic of cytomegalovirus in the community. However, there is no evidence to suggest that cytomegalovirus ever behaves in an epidemic fashion, 9 and further, virus was detected throughout the entire year the study was carried out. Crowding and living in depressed socioeconomic conditions have been shown to facilitate dissemination of cytomegalovirus, at least when measured by the presence of viruria? ~ Such conditions are present for the majority of patients studied here but certainly to no more degree than that of the usual large outpatient population in the urban area. T h e obvious factor to be considered for the population studied here is its residence in a tropical climate. We know of no other large study of respiratory tract viruses in a tropical climate using methods (e.g., human fibroblast cultures) sensitive for the detection of cytomegalovirus. Thus it may be that environmental conditions as they exist in Bangkok (relative humidity 70 to 87 per cent, monthly mean temperature between 28.0 to 32.0 ~ C. with a minimum rarely below 25.0 ~ C.) and attendant customs of daily life in some way play a major role contributing to the much greater incidence of respiratory tract infection with cytomegalovirus, as compared to populations in temperate climates. Serologic findings on patients positive for cytomegalovirus indicate the rarity with which Complement fixation antibody is present during active infection of the respiratory
Excretion of cytomegalovirus
503
tract. This could be anticipated since even in instances of systemic cytomegalovirus infection where the amount of antigenic stimulus is presumably much greater complement fixtion antibody is very slow to appear, although macroglobulin and neutralizing antibody appear much earlierS, 15 However, most patients excreting cytomegalovirus did have some evidence of circulating antibody as measured by neutralization tests, fluorescent anticytomegalovirus g a m m a globulin a n d / o r IgM. The significance of wide circulation of cytomegalovirus in this population is unknown in terms of morbidity. Though data are not available, it is the impression of local physicians that cytomegalic inclusion disease of newborn infants is a rarely encountered condition. I t may well be that widespread circulation of virus at such an early age renders the vast majority of girls immune well before child-bearing age so that transplacental infection of the fetus occurs only infrequently. REFERENCES 1. Farber, S., and Wolback, S. B.: Intranuclear and cytoplasmic inclusions ("protozoan-like bodies") in the salivary glands and other organs of infants, Amer. J. Path. 8: 123, 1932. 2. Nichol, K. P., and Cherry, J. D.: Bacterialviral interrelationships in respiratory infections of children, New Eng. J. Med. 277: 667, 1967. 3. Weller, T. H., and Rowe, W. P.: The human cytomegaloviruses, in Diagnostic procedures for viral and rickettsial diseases, ed. 3, Lennette and Schmidt, editors, New York, 1964, American Public Health Association, Inc., p. 7O7. 4. Lang, D. J., and Noren, B.: Cytomegaloviremia following congenital infection, J. PEDmT. 73: 812, 1968. 5. ttanshaw, J. B., SteinfeId, H. J., and White, C. J.: Fluorescent antibody test for cytomegalovirus macroglobulin, New Eng. J. Med. 279: 566, 1968. 6. Lewis, V. J., Jones, W. L., Brooks, J. B., and Cherry, W. B.: Technical considerations in the preparation of fluorescent-antibody conjugates, Appl. Microbiol. 12: 343, 1964. 7. Olson, L. C., and Lexomboon, U.: Unpublished observations. 8. Glezen, W. P., Wulff, H., Lamb, G. A., Ray, C. G., Chin, T. D. Y., and Wenner, It. A.: Patterns of virus infections in families with
5 04
Olson et al.
acute respiratory illnesses, Amer. J. Epldem. 86: 350, 1967. 9. Starr, J. G., and Gold, E.: Screening of newborn infants for cytomegalovirus infection, J. PEDIAT. 73: 820, 1968. I0. Li, F. P., and Hanshaw, J. B.: Cytomegalovirus infection among migrant children, Amer. J. Epidem. 86: 137, 1967.
The ]ournal o] Pediatrics September 1970
11. Lang, D. J., and Hanshaw, J. B.: Cytomegalovlrus infection and the post-infusion syndrome: Recognition of primary infections in four patients, New Eng. J. Med. 280: 1145, 1969.
P E D I A T R I C S DerrickB. Jelliffe,
Editor
Respiratory tract excretion of cytomegalovirus in Thai children Cytomegalovirus has been found to be commonly present in the respiratory tract secretions of Thai children. Eleven per cent of children with pertussis syndrome, 18.1 per cent of 342 children with respiratory tract infections, and 15.1 per cent of well babies were positive [or cytomegalovirus. Virus was most commonly present in children less than one year of age, the incidence falling progressively with age so that it was unusual to recover cytomegalovirus #ore children over the age of 4 years. Most patients tested possessed serum neutralizing and immunoglobulin M antibody at the time virus was being excreted, but few had complement-fixing antibody.
Lloyd C. Olson, M.D., R a m p e y a Ketusinha, M.D.,
Pethai Mansuwan, M.D., and Rapin Snitbhan, M.D. BANGKOK,
THAILAND
A L W~ O U a H T H E R E is considerable evidence to indicate that cytomegalovirus infection in m a n is a common occurrence, little is known as to how and when individuals of a population become infected. In particular, the role of excretion of cytomegalovirus from the respiratory tract as a means of transmission remains unknown. Salivary gland infection has been demonstrated repeatedly, 1 although the rarity with which this agent has been recovered during virologic studies of respiratory tract flora might suggest that inFrom the Department of Virology, S E A T O Medical Research Laboratory; Children's Hospital; and Faculty of Public Health, Mahidol University. Rtpr~nt address: L. C. Olson, USA Medical Component SEATO, APO San Francisco, Calif. 96346.
fectious virus does not gain access to the environment from this site. 2 During a longitudinal study of the respiratory viruses of children living in Bangkok, an unexpected number yielded cytomegalovirus from the respiratory tract. This report details isolation rates of this virus from various groups of children living in this tropical locale. M A T E R I A L AND M E T H O D S Patient population. Children from whom nasopharyngeal swabs were obtained were part of 3 groups at the ChiIdren's Hospital, Bangkok, being studied for respiratory viruses. Those with upper respiratory infections were selected for study by a single VoI. 77, No. 3, pp. 499-504
500
Olson et al.
physician in the outpatient department if they were under the age of 12 years and had been ill less than 48 hours with fever and respiratory tract symptoms. Patients who had clinical symptoms consistent with the diagnosis of whooping cough of less than 3 weeks' duration were studied as a separate group. Infants in the newborn nursery were arbitrarily selected for study. Well children were selected from the well baby clinic if, by history and physical examination, they were free of symptoms. Virologic procedures. Nasopharyngeal swabs were obtained and immediately placed in 2.0 ml. of Hanks balanced salt solution containing 0.4 per cent bovine plasma albumin. Urine was collected from newborn infants and centrifuged lightly to remove debris. All specimens were treated with antibiotics (penicillin-streptomycin-neomycin) and 0.1 ml. was inoculated into each of 3 tube cultures of WI-38 cells (originally obtained from Dr. Leonard Hayflick, Wistar Institute). After absorption overnight at 37 ~ C., media were decanted and replaced with medium L-15 containing 2 per cent heatinactivated fetal bovine serum and antibiotics (penicillin-streptomycin-amphotericin B). Cultures were thereafter incubated at 33 ~ C., observed microscopically for evidence of cytopathic effects twice weekly, and fed with fresh maintenance media at intervals of 1 to 2 weeks. Cultures were considered negative only if cell monolayers were maintained in satisfactory condition for at least 6 weeks without any evidence of focal cytopathic effects. Virus identification. Cytomegalovirus was presumptively identified on the basis of cytopathic effect characteristics, as described by others, a and usually appeared in the second to fourth week after inoculation. Cytopathic effect was allowed to progress until it involved most of the cell sheet or until cells otherwise began to spontaneously degenerate, at which times cells were scraped into the media, disrupted by freezing and thawing rapidly 3 times, centrifuged, and the supernatant passed to fresh cell cultures. On passage the belated reappearance of char-
The Journal of Pediatrics September 1970
acteristic cytopathic effects was taken as confirmatory evidence of the presence of cytomegalovirus. Some procedures were admittedly not those conventionally employed for cytomegalovirus, but originally these were designed as a detection system for rhinoviruses. When it became obvious that cytomegalovirus was frequently present, a series of 78 specimens from the group of children with upper respiratory infections were inoculated separately into 2 groups of cell cultures and incubated at 33 and 37 ~ C., respectively. While cytopathic effects appeared more slowly at 33 ~ C., the same 16 specimens were ultimately positive in both groups. Thus, in order to maintain a uniform procedure, incubation at 33 ~ C. was continued. In no instance did a primary isolate of cytomegalovirus fail to pass after freeze-thaw. O n occasion, adenovirus cytopathic effect was initially mistaken for the cytopathic effect of cytomegalovirus. The much more rapid growth in WI-38 ceils and the ability to produce cytopathic effects in human heteroploid cell lines then made adenoviruses easily recognizable. Complement fixation tests. Complement fixation antibody was determined using, in separate tests, both an antigen prepared commercially from AD-169 strain (Microbiological Associates, Rockville, Md.) and from a locally isolated strain using a method similar to that of Lang and Noren. 4 Tests were carried out in disposable microtiter plates using 2 to 4 units of antigen with overnight fixation. Neutralizing antibody tests. Screening tests for serum neutralizing antibody on heat-inactivated serum (56 ~ C., 30 minutes) were carried out at a serum dilution of 1:4 by mixing with approximately 100 tissue culture 50 per cent doses (TCIDs0) of one of the strains isolated in this study (strain No. 154). After reacting at room temperature for one hour, the mixture was inoculated into replicate tube cultures of WI-38 cells. Tests were read weekly for 4 weeks. Consistently obvious differences in the amount of cytopathic effect in the test specimens in
Volume 77 Number 3
Excretion o[ cytornegalovirus
Table I. Recovery of cytomegalovirus by age groups in patients with upper respiratory infections Age
< l yr. 1
2 3 >4
Totals
No. cultured
No. positive
81 86 51 37 87 342
32 17 6 5 2 62
Table ]I. Recovery of cytomegalovirus by age groups in patients with pertussis syndrome No. cultured
No. positive
%
Age
39.5
<1 yr.
12
4
19.8
1
19
3
11.8 13.5 2.3 18.1
2 3 >4 Total
14 12 43 100
2 I 1 11
contrast to that in the control specimens was considered as evidence of antibody. Indirect immunofluorescent antibody tests. In the tests for fluorescent antibody a local virus strain (No. 154) was employed; the method followed that of Hanshaw and associates 5 with the following modifications. Tests were carried out using separately goat antihuman gamma globulin and goat antihuman IgM (Hyland Laboratories, Los Angeles, Calif.) after conjugation with fluorescein isothiocyanate. 6 Sera to be tested were diluted 1:10 in phosphate-buffered saline p H 7.2 and adsorbed for 2 hours at room temperature on tube monolayers of WI-38 cells previously washed 3 times with phosphate buffered saline. Fluid was then aspirated and tested without further treatment. This procedure was found to remove effectively nonspecific immunofluorescence from most sera. Goat sera were adsorbed with WI-38 cell suspensions. ~ RESULTS
In the group of children studied with upper respiratory tract infections, of the approximately 45,000 children seen with this diagnosis in the clinic of Children's Hospital, 342 were studied from January to December, 1968. From this group, cytomegalovirus was isolated from 62 patients, or 18.1 per cent of those studied. Forty of the patients were boys and 22 were girls. Virus was isolated most frequently from children less than one year of age, the per cent positive progres-
50 1
%
33.3 16.0 14.3 8.3 2.3 11.0
slvely dropping with increasing age so that it was unusual to recover virus after the age of 4 years (Table I). Similar recovery rates and age distribution of patients who were positive for cytomegalovirus were found during the period from July, 1968, to January, 1969, in 100 patients with a clinical diagnosis of whooping cough (Table II). In this group, 8 of the cytomegalovirus positive patients were girls and 3 were boys. Patients in the group with upper respiratory infections were also studied for the presence of other respiratory virusesY From the 62 patients excreting cytomegalovirus, another virus was isolated 17 times, while cultures of 91 of the 280 cytomeglovirusnegative patients were positive. With the exception of a rubella epidemic early in the year and the appearance of the 1968 A2 influenza variant in September, there were no unusual features of respiratory diseases throughout this year as compared to other years in the community. Virus was recovered from nasopharyngeal swabs from 10 of 66 well children under the age of one year studied during the period from August to November, 1968. This suggests a lower frequency of virus excretion in asymptomatic children, but the smaller size of this group and dissimilarity of age composition by month renders further analysis impossible (Table III). To rule out the unlikely possibility that virus recovered represented congenitally acquired infection, 187 urine samples were
502
Olson et al.
The Journal o] Pediatrics September t970
Table I l L Cytomegalovirus-positive patients, by month of age
Age (too.)
No. positive/No, cultured Upper respiratory infections group Well babies
1
2 3 4 5 6 7 8 9 10 11
1/I 1/2 4/7 9/13 6/17 2/11 7/17 2/13 32/81
0/6 1/12 4/11 1/10 2/10 I/2 1/5 O/5 O/4 0/I 10/66
collected from randomly selected newborn infants in July and October, 1968. Only 2 were positive for virus. Paired sera (acute phase and 2 weeks later) were collected from children in the upper respiratory infections and pertussis study groups. Thirty-four pairs from the upper respiratory infections group and 9 from the pertussis group, all from cytomegalovirus excretors, were tested (Table I V ) . Testing of the acute sera showed complement fixation antibody in sera of 4 of 9 pertussis patients, and 5 of tile 34 in the upper respiratory infections group. However, sera obtained 2 weeks later showed no increase in titer in sera from the latter group, while 2 of the pertussis patients had fourfold increases in complement fixation antibody, No differences in reactivity between the local strain and AD-169 were were found. Neutralizing antibody was present in most acute phase sera. Of the 38 sera demonstrating neutralizing antibody, 37 were confirmed by the demonstration of anticytomegalovirus globulin by immunofluorescence. Of these 37, anticytomegalovirus I g M was present in 26 of the sera at the dilution
Table IV. Antibody determinations in cytomegalovirus-positive patients
Determinations
Total tested Neutralizing antibody pos. FAT: globulin poe. FAT: IgM poe, Total CF poe, CF pos. IgM FAT pos.
Upper respiratory infections Pertussis group group
34 30 29 24
9 8 8 2
5 0
7 0
Total CF neg. 29 2 CF neg. IgM pos. 21 2 FAT = fluorescent antibody te~t. CF = complementfixing antibody.
tested. None of the sera which were complement fixation positive were also positive for IgM. Four acute phase sera were negative in all tests. All sera positive by neutralization tests were also positive by at least one other method; one serum was positive only for IgM. DISCUSSION Surveys for the presence of cytomegalovirus in the respiratory tracts of children in Bangkok have shown the virus to be commonly present, having been detected in 21.2 per cent of 378 children less than 4 years of age. Recovery rates were highest from infants less than one year of age: 40 per cent of those with upper respiratory infections, 33 per cent with a pertussis syndrome, and 15 per cent of well babies. T h e high frequency with which virus was recovered from young children in a single sample implies virus is present over a prolonged period of time, although serial follow-up will be necessary for confirmation. Although cytomegalovirus was recovered more frequently from children with upper respiratory infections, there is no evidence to suggest its presence was related to symptoms. In fact the isolation of other respiratory viruses
Volume 77 Number 3
from 30 per cent of the patients, irrespective of whether they were shedding cytomegalovirus or not, would argue against this. Other studies of viruses from the respiratory tract s, s have rarely or never encountered cytomegalovirus in children. It is doubtful if this is simply because the virus was not detected in other studies since, in the present study, many specimens were positive even within the usual observation period of 2 weeks. Another possibility explaining the high incidence might be that the current study coincided with an epidemic of cytomegalovirus in the community. However, there is no evidence to suggest that cytomegalovirus ever behaves in an epidemic fashion, 9 and further, virus was detected throughout the entire year the study was carried out. Crowding and living in depressed socioeconomic conditions have been shown to facilitate dissemination of cytomegalovirus, at least when measured by the presence of viruria? ~ Such conditions are present for the majority of patients studied here but certainly to no more degree than that of the usual large outpatient population in the urban area. T h e obvious factor to be considered for the population studied here is its residence in a tropical climate. We know of no other large study of respiratory tract viruses in a tropical climate using methods (e.g., human fibroblast cultures) sensitive for the detection of cytomegalovirus. Thus it may be that environmental conditions as they exist in Bangkok (relative humidity 70 to 87 per cent, monthly mean temperature between 28.0 to 32.0 ~ C. with a minimum rarely below 25.0 ~ C.) and attendant customs of daily life in some way play a major role contributing to the much greater incidence of respiratory tract infection with cytomegalovirus, as compared to populations in temperate climates. Serologic findings on patients positive for cytomegalovirus indicate the rarity with which Complement fixation antibody is present during active infection of the respiratory
Excretion of cytomegalovirus
503
tract. This could be anticipated since even in instances of systemic cytomegalovirus infection where the amount of antigenic stimulus is presumably much greater complement fixtion antibody is very slow to appear, although macroglobulin and neutralizing antibody appear much earlierS, 15 However, most patients excreting cytomegalovirus did have some evidence of circulating antibody as measured by neutralization tests, fluorescent anticytomegalovirus g a m m a globulin a n d / o r IgM. The significance of wide circulation of cytomegalovirus in this population is unknown in terms of morbidity. Though data are not available, it is the impression of local physicians that cytomegalic inclusion disease of newborn infants is a rarely encountered condition. I t may well be that widespread circulation of virus at such an early age renders the vast majority of girls immune well before child-bearing age so that transplacental infection of the fetus occurs only infrequently. REFERENCES 1. Farber, S., and Wolback, S. B.: Intranuclear and cytoplasmic inclusions ("protozoan-like bodies") in the salivary glands and other organs of infants, Amer. J. Path. 8: 123, 1932. 2. Nichol, K. P., and Cherry, J. D.: Bacterialviral interrelationships in respiratory infections of children, New Eng. J. Med. 277: 667, 1967. 3. Weller, T. H., and Rowe, W. P.: The human cytomegaloviruses, in Diagnostic procedures for viral and rickettsial diseases, ed. 3, Lennette and Schmidt, editors, New York, 1964, American Public Health Association, Inc., p. 7O7. 4. Lang, D. J., and Noren, B.: Cytomegaloviremia following congenital infection, J. PEDmT. 73: 812, 1968. 5. ttanshaw, J. B., SteinfeId, H. J., and White, C. J.: Fluorescent antibody test for cytomegalovirus macroglobulin, New Eng. J. Med. 279: 566, 1968. 6. Lewis, V. J., Jones, W. L., Brooks, J. B., and Cherry, W. B.: Technical considerations in the preparation of fluorescent-antibody conjugates, Appl. Microbiol. 12: 343, 1964. 7. Olson, L. C., and Lexomboon, U.: Unpublished observations. 8. Glezen, W. P., Wulff, H., Lamb, G. A., Ray, C. G., Chin, T. D. Y., and Wenner, It. A.: Patterns of virus infections in families with
5 04
Olson et al.
acute respiratory illnesses, Amer. J. Epldem. 86: 350, 1967. 9. Starr, J. G., and Gold, E.: Screening of newborn infants for cytomegalovirus infection, J. PEDIAT. 73: 820, 1968. I0. Li, F. P., and Hanshaw, J. B.: Cytomegalovirus infection among migrant children, Amer. J. Epidem. 86: 137, 1967.
The ]ournal o] Pediatrics September 1970
11. Lang, D. J., and Hanshaw, J. B.: Cytomegalovlrus infection and the post-infusion syndrome: Recognition of primary infections in four patients, New Eng. J. Med. 280: 1145, 1969.