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Pharyngeal colonization with Haemophilus influenzae type b in children in a day care center without invasive disease
Pharyngeal colonization with Haemophilus influenzae type b in children in a day care center without invasive disease During an 18-month period, monthly pharyngeal cultures for Haemophilus influenzae type b (Hib) were obtained from 66 children and their caretakers in a day care center in which no systemic disease caused by Hib occurred. The average colonization rate for Hib was 10%, and ranged from 0% to 23% for a single month. Infants housed in a separate building with a cohorted staff were not colonized by Hib. However, 71% o f the toddler group and 48 % o f the preschool group became colonized by Hib at some time during the 18-month-study. 0 f 8 9 Hib isolates, 93% were biotype 1 (Kilian), and 90% o f these had a similar outer membrane protein profile, designated subtype 1L. This strain was recovered from children at the center for 15 o f 18 months. No invasive disease occurred. Thus, Hib may be widespread among preschool children in a day care center and persist for longer than a year without resulting in systemic disease. (J PEDIATR 106:712, 1985)
T. V. Murphy, M.D., Dan Granoff, M.D., D. F. Chrane, R.N., K. D. Olsen, B.S., S. J. Barenkamp, M.D., S. F. Dowell, and G. H. McCracken, Jr., M.D. Dallas, Texas, and St. Louis, Missouri
PHARYNGEAL COLONIZATION by H a e m o p h i l u s influenzae type b is present in fewer than 5% of healthy preschool children not exposed to a patient with systemic disease? ,2 By contrast, carrier rates as high as 50% have been found in preschool children who attend day care centers in which there was one or more individuals with Hib disease?' ~ Limited information is available describing the natural history of Hib colonization among children in day care in the absence of invasive disease. In 1963, Turk 5 reported that nine (11%) of 85 healthy preschool children in a day nursery without disease were colonized by Hib. More recently, surveys of children insix day care centers From the Department of Pediatrics, Southwestern Medical School, The University o f Texas Health Science Center, Dallas; and the Edward Mallinckrodt Department o f Pediatrics, Washington University School o f Medicine, and the Division o f Infectious Diseases, St. Louis Children's Hospital. Supported in part by the Zale Foundation and by Grant AI 17572 from the National Institute o f Allergy and Infectious Diseases. Submitted for publication July 9, 1984; accepted Sept. 21, 19841 Reprint requests: Trudy Murphy, M.D., Research Assistant Professor o f Pediatrics, The University o f Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235.
712
The Journal o f P E D I A T R I C S
where there was no H a e m o p h i l u s disease found only 3% (six of 223) colonization.6-8 We prospectively observed for 18 months well children who were enrolled in a day care center in Dallas and in whom there had been no known systemic H a e m o p h i l u s disease. Our objective was to determine Hib colonization in well children enrolled in day care who had no exposure to a patient with H a e m o p h i l u s disease. By using the strain Hib SDS-PAGE
Haemophilus influenzae type b
Sodium dodecyl sulfat~polyacrylamide gel eleetrophoresis
markers provided by biotyping and outer membrane protein gel analysis, we were able to identify specific strains colonizing children in the center.
METHODS The day care center studied was selected because, to the knowledge of the director, no child had been hospitalized with' invasive Hib disease in its 29-month history. The center was privately owned and operated for profit. Children came from families with a wide range of educational, economic, geographic, and ethnic backgrounds. Total
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enrollment during the study ranged from 80 to 100 children. Attendees were grouped by age. Infants (aged 1.5 to 17 months) were cared for by a single caretaker in a separate building. Toddlers (aged 18 to 35 months), a preschool group (36 to 71 months), and children who attended after school (5 to 8 years) were housed in different rooms of a second building and shared a common play yard. Children in the preschool group were further subdivided for learning activities approximately 2 hours each day. Meals were prepared by a cook and were eaten in the assigned classrooms. Children were promoted from one group to the next throughout the year after attaining the next age level. Infants had no contact with older groups except during the 2 weeks before promotion. At this time, an infant spent 2 hours a day with the toddlers. Study design. Parents of children at the day care center were asked to participate in the study by the center director. Informed, written consent was obtained for a single, monthly pharyngeal culture from each participant. The investigators made two visits to the center each month to obtain the cultures. N O change was made in the existing child care and educational programs. The study was conducted between December 1980 and May 1982. Absences related to illness were monitored by the center director and reported to the investigators. Illness consistent with invasive Hib disease or resulting in hospitalization of a child was investigated. We did not monitor minor illnesses or antibiotic usage. During March, 1981, 10 children with active rhinorrhea were observed during play time to determine possible modes of exchange of nasal secretions. Cultures of nasal secretions were obtained from the upper lip of three colonized children, on three occasions spaced by at least 1 day. These children also were asked to cough onto a chocolate agar plate placed an inch from the mouth. Pharyngeal cultures. Cultures were obtained by thorough swabbing of both tonsils and the posterior pharynx. Swabs were placed in brain-heart infusion broth supplemented with 5% Levinthal base and 5 U / m l bacitracin for overnight incubation at 37 ~ C in 5% CO2. Aliquots of overnight broth were plated onto chocolate and Hib antiserum agar, prepared as previously described.9 Plates were incubated in 5% CO2 at 37 ~ C for 24 hours and examined for colonies with typical morphology on chocolate agar or precipitin halos on antiserum agar. ~~ This method provided rapid screening of large numbers of samples and identification of Hib organisms present in low concentrations. H. influenzae was identified by growth requirements for X and V factor. H Capsular typing was confirmed by slide agglutination with commercial Hib antiserum (Difco Laboratories, Detroit). Isolates were assigned to one of six biotypes using the method of Kilian. H
a . influenzae type b infection in day care centers
71 3
The outer membrane protein patterns of Hib isolates were examined by SDS-PAGE using two gel systems. ~2 Subtype designation was assigned using the results of both gels) 3 Outer membrane protein subtypes were determined for H i b isolated from each child's first and last positive culture. In addition, all Hib isolates from four children who had multiple positive cultures were examined by these ~techniques. In each of these children consecutive isolates showed the same respective outer membrane protein subtype.. In the remaining children, if first and last isolates were identical by biotype and outer membrane protein subtype, respectively, then Hib isolated from consecutive intervening months were tested only for biotype. If concordant, these isolates were presumed to have the same outer membrane protein subtype a s the first and last isolates for calculation of the duration of colonization. For purposes of our study, two Hib organisms were considered to be the same strain if the respective biotypes and outer membrane protein subtypes were identical. The incidence of Hib colonization was defined as the number of children with at least one positive culture for Hib divided by the number of children participating in the study. The duration Of colonization was defined as the number of months of positive cultures for a given strain of Hib. When positive cultures were obtained both before and after one or two negative cultures, the negative cultures were counted as if positive for this calculation. Acquisition of Hib was defined as a positive culture after three negative cultures. Positive cultures during the first 3 months of the study were considered new acquisitions. RESULTS Culture specimens were obtained i n 83 children and 13 day care staff during the 18-month study. Data from 66 children and 12 staff who participated for >--5 months are presented. This subpopulation comprised between 40% and 50% of the center's total monthly enrollment, was similar in demographic features in the total population, and is referred to as the study population. Sixty-one percent of study children were white, 26% were black, 12% were Hispanic, and 2% were Asian. Fifty-three percent were boys. Eleven sets of siblings participated in the study, and three caretakers had a total of four of their own children enrolled in the center. Subjects remained enrolled in the study for a mean of 11.8 months (median 10 months) (Table I). Twenty-five children participated for the entire 18 months. Accretion and attrition rates of subjects participating and leaving the study averaged 1.8 and 1.2 subjects per rtrbnth, respectively. Thirty-two percent of study children were promoted to the next older age group while participating, including seven of 17 infants, 14 of 21 toddlers, and none of 28 pre-
7 14
Murphy et al.
25
The Journal of Pediatrics May 1985
84
zo = '15
40 e
s
z
T P T P T P T P T P T P Dec-Feb Mor-Moy Jun-Aug Sept-Nov Dec-Feb Mar-MQy '80 '8t '82
Figure. Quarterly prevalencerate of Haemophilus influenzae type b, subtype 1L pharyngeal colonization in children assigned to toddler and preschool groups. At least one pharyngeal culture was obtained from each child during calendar quarter. Absolute number of children assigned to toddler group decreased after third quarter of study, whereas preschool group increased in number. Hib subtype 1L was not isolated during last quarter of study. Table I. Number of months of study participation by group assignment
Group
Age (mo)
Number tested*
Months of follow-up (mean ++_SD)
Infant Toddler Preschool After-school Staff
1.5 to 17 ! 8 to 35 36 to 71 >72 Adult
17 28 35 7 12
6.6 • 315 8.8 • 4.6 8.9 • 5.3 12.7 • 6.0 12.8 _+ 3.0
*In this analysis, children promoted from one age group to another were counted in both groups.
Table II. Acquisition of pharyngeal colonization with Haemophilus influenzae type b by children and staff in a day care center
Group
Number cultured
Number positive
Incidence*
Infant Toddler Preschool After-school Staff
17 21 21 7 12
1t 15 10 1 1
6 71 48 14 8
*Number of children with at least one culture positive for Hib per 100 children in the center during 18 months of study, "~Hib colonization acquired after promotion to toddler group.
and after-school children. Fourteen of 17 infants enrolled after the study began. Hib colonization. During the 18 months, 750 cultures were obtained from children, and 154 from staff. Eightynine (10%) cultures were positive for Hib. The prevalence rate of colonization was 7% in the first month of the study,
and was greatest 4 months later (23% in March 1981). The rate was 0% during the last 2 months of the study. Seventy-one percent and 48% of children assigned to the toddler and preschool groups, respectively, had at least one positive culture for HIB during 18 months (Table II). Acquisition of Hib colonization was greatest during the first 6 months (December to May) of the study, averaging 2.2 children per month in the toddler group (13 of 17 children) and 1.2 children per month in the preschool group (seven of 16). During the last 12 months, the rate of acquisition of Hib was lower, averaging 0.25 child per month (six of 28) for children assigned to either group. Of the seven infants who were promoted to the toddler group during the study, only one became colonized by Hib. This infant was the only one transferred during the first 6 months of the study, when acquisition rates were highest. Colonization lasted for an average of 2.4 months per acquisition (median 1.0 month, range 1 to 7 months). The mean duration of colonization for toddlers was similar to that for children in the preschool group (2.6 a n d 2.3 months, respectively). Isolate characterization. The biotype of the 89 Hib strains isolated during the study fell into five of six groups described by Kilian. However, 93% of isolates were biotype 1. Eighty-four percent of acquisitions with biotype 1 organisms were associated with isolates of the outer membrane protein subtype 1L. The remaining isolates were either subtype 1H (13%) or 2L (3%). The prevalence of colonization by 1L organisms in the toddler and preschool groups, by calendar quarter, is shown in the Figure. This strain was the predominant isolate at the center for 15 of 18 months, and was the only strain that colonized children in all groups except the infants. It appeared to have been present initially in the toddlers, in which it became most prevalent, and then to have spread to the preschool children. Subtype 1H Hib was isolated from four children in the preschool group. One child was colonized for 3 months at the beginning of the study, and three others had positive cultures for 1 month only during March 1982. These three children previously had been carriers of 1L strains for durations of 2 to 6 months. Hib subtype 2L was isolated from a single child in the preschool group on three occasions over 8 months. Nasal secretions in toddlers. Cultures of nasal secretions from the upper lip of three toddlers who previously had been identified as Hib carriers werepositive for Hib seven of nine times over a 6-day period. Negative cultures resulted when nasal secretions had dried. Hib was not recovered from any of the "cough" plates held 1 inch from the mouth of five toddlers colonized by Hib. Ten children with rhinorrhea who were assigned to the
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toddler group were selected for observation of the handling of their nasal secretions. Total observation time was 150 minutes. Wiping or picking the nose, followed by placing the "contaminated" hand on a toy or other inanimate object occurred six times during this period. Coughing or sneezing occurred twice. Caretakers cleaned the rhinor 2 rhea with paper tissues three times but did not wash their own hands or the hands of the children. Nasal secretions appeared to be spread most efficiently at meals, an activity not included in the planned observations. Children shared food (mixed with nasal secretions) by taking a handful of food from their own mouth and placing it into the next child's mouth. On one occasion, an adult caretaker was observed to clean the faces of all toddlers with a single moist washrag. No invasive Hib disease was identified in study subjects or in members of their families during the 18 months. One child in the preschool group received treatment as an outpatient for pneumonia of undetermined cause during July 1981. Hib 1L had been isolated from the pharynx of this child in December 19801 but not during the intervening 7 months. Another infant who had left the day care center 2 months after the study began (and who therefore was not included in the study) developed osteomyelitis 9 months later from an Hib isolate with the 1H subtype. Hib had not been isolated from the pharynx of this child while she was at the center. DISCUSSION We have demonstrated that children in a day care center selected only because there were no proven cases of disease had high rates of acquisition and colonization by type b H. influenzae. High rates of colonization have been observed previously among children in day care, but nearly always in children exposed to a patient with type b Haemophilus disease. Recently, point prevalence studies at six centers without disease demonstrated relatively low rates of colonization by Hib (0% to 7%), 68 similar to those found in children who do notattend day care (1.4%). 2 HOwever, it is possible that higher rates would be found among children in day care if the children were observed for longer periods. At the Frank Porter Graham Development Center, where colonization has been monitored for more than a decade in the absence of disease, fewer than 5% of children were colonized by Hib for 87% of the months from 1970 to 1980. However, colonization was 5% to 10% for 11% of months, and between 10% and 20% for 1.6% of months during this period (Dr. Floyd Denny, University of North Carolina, Chapel Hill, personal communication, 1984). In our study, a single strain of Hib (1L) was found to colonize 67% of toddler and 33% of preschool group children, and to persist during 15 of the 18 months that
1t. influenzae type b infection in day care centers
715
cultures were obtained. The Hib subtype 1L strain was probably introduced into the center before the study began, possibly into the toddler group. Colonization of two children assigned to the toddler group (and the mother of one of them) was demonstrated in the first culture survey. Persistence of Hib colonization has been reported among contacts of individuals with disease in day care for np to 14 months. ~4 In our study center, without cases of invasive disease, Hib 1L was present in some children for 15 months. It is not known whether persistence of Hib among these children was related to characteristics of the particular Hib strain or to other factors (e.g., physical characteristics of the center, behavior patterns of the children, intercurrent viral infection). Three children who were carriers of Hib 1L were later Colonized by Hib 1H for a shorter period. However, it is possible that these children were colonized simultaneously by more than one strain. Dual coloniza~km would have gone unrecognized, because single colonies were selected for storage and characterization. Nevertheless, the second strain was detected only after one to three negative cultures, suggesting that colonization by Hib organisms with the 1H subtype represented new acquisitions. The lack of invasive disease among our study population is of particular interest, considering the high acquisition rates of potentially pathogenic Hib strains. When grown on solid media, all of the organisms were mucoid and formed strong precipitin halos on Hib antiserum agar? ~ Hib organisms with the 1L, 1H, or 2L subtypes have all been associated with invasive disease? z In a recent survey, 1L organisms accounted for only 7% of isolates from 256 patients hospitalized in 22 states~3; nevertheless, isolates with this subtype once may have been the predominant disease-producing strains in the United States. ~5 Furthermore, Hib organisms with the 1L subtype were isolated from four patients during a day care center outbreak of Hib disease within the same metropolitan area while our study was in progress. ~6 One possible reason for the lack of invasive disease in our study was the failure of Hib to colonize the youngest children, who would have been at greatest risk for developing disease, by virtue of their lack of immunity.17In the day care center studied, infants were isolated physically in their own building, and few infants were promoted to the toddler group during the early months of the study, when acquisition rates were greatest. In addition, the total number of infants studied was small. Colonized children at the center were recognized only because cultures were taken for the sttttty. There are limited data to suggest that healthy children with random cultures positive for Hib are not at increased risk for disease? For the present, prophylactic treatment with rifampin would not appear to be indicated for children who
7 16
Murphy et al.
are incidentally found to be H i b carriers, in the absence of exposure to a patient with Haemophilus disease. F r o m our results, it is suggested t h a t child day care provides a potential environment for high rates of acquisition of Hib, even in the absence of invasive disease. Acquisition of Hib colonization is associated with developm e n t of large concentrations of systemic antibody, 8.~7 conferring i m m u n i t y against disease. However, determining the actual risk of developing invasive disease r a t h e r t h a n i m m u n i t y will require a larger, prospective study. F u r t h e r research is necessary to define those f a c t o r s - microbial, host, or e n v i r o n m e n t a l - - t h a t m a y contribute to development of the invasive disease in a given population. We thank Mrs. Elaine Krause, director of the study day care center, for allowing the study to take place and for obtaining informed consent from parents of enrolled children; Mrs. Teresa Zweighaft for laboratory assistance during the first 6 months of the project; Marian Troup for preparation of the manuscript; and Katherine McCullough for technical assistance. J. B. Robbins, M.D., Bureau of Biologics, Bethesda, Maryland, provided hyperimmune burro anti-Hib serum. REFERENCES
1. Michaels RH, Poziviak CS, Stonebraker FE, Norden CW: Factors affecting pharyngeal Haemophilus influenzae type b colonization rates in children. J Clin Microbiol 4:413, 1976. 2. Hampton CM, Barenkamp S J, Granoff DM: Comparison of outer membrane protein subtypes of Haemophilus influenzae type b isolates from healthy children in the general population and from diseased patients. J Clin Microbiol 18:596, 1983. 3. Granoff DM, Daum RS: Spread of Haemophilus influenzae type b: Recent epidemiologic and therapeutic considerations. J PEDIATR 97:854, 1980. 4. Barenkamp S J, Granoff DM, Munson RS Jr: Outermembrane protein subtypes of Haemophilus influenzae type b and spread of disease in day care centers. J Infect Dis 144:210, 1981. 5. Turk DC: Naso-pharyngeal carriage ofHaemophilus influenzae type b. J Hyg Camb 61;247 , 1963.
The Journal of Pediatrics May 1985 6. Ward JL, Gorman G, Phillips C, Fraser DW: Haemophilus influenzae type b disease in a day care center: Report of an outbreak. J PEDIATR 92:713, 1978. 7. Gran0ff DM, Gilsdorf J, Gessert C, Basden M: Haemophilus influenzae type b disease in a day care center: Eradication of carrier state by rifampin. Pediatrics 63:397, 1979. 8. Cox F, Trincher R, Rissing JP, et al: Rifampin prophylaxis for contacts of Haemophilus influenzae type b disease. JAMA 245:1043, 1981. 9. Murphy TV, Chrane DF, McCracken GH Jr, Nelson JD: Rifampin prophylaxis versus placebo for household contacts of children with Haemophilus influenzae type b disease. Am J Dis Child 137:627, 1983. 10. Michaels RH, Stonebraker FE, Robbins JB: Use of antiserum agar for detection of Haemophilus influenzae type b in the pharynx. Pediatr Res 9:513, 1975~ 11. Kilian M: Haemophilus. In Lanette EH, editor: Manual of clinical microbiology, ed 2. Washington, D.C., 1980, American Society for Microbiology, pp 330-336. 12. Barenkamp SJ, Munson RS Jr, Granoff DM: Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. J Infect Dis 143:668, 1981. 13. Granoff DM, Barenkamp SJ, Munson RS Jr: Outer membrane protein subtypes for epidemiologic investigation of Haemophilus influenzae type b disease. In Sell SH, Wright PF, editors: The biology of Haemophilus influenzae. New York, 1982, Elsevier Science Publishing Co., pp 43-55. 14. Ginsburg CM, McCracken GH Jr, Rae S, Parke JC Jr: Haemophilus iJ~uenzae type b disease: Incidence in a day care center. JAMA 238:604, 1977. 15. Barenkamp S J, Granoff DM, Pittman M: Outer membrane protein subtypes and biotypes of Haemophilus influenzae type b: Relation between strains isolated in 1934-1954 and 1977-1980. J Infect Dis 148:1127, 1983. 16. Murphy TV, McCracken GH Jr, Moore BS, et al: Haemophilus influenzae type b disease after rifampin prophylaxis in a day care center: Possible reasons for its failure. Pediatr Infect Dis 2:193, 1983. 17. Granoff DM, Gilsdorf J, Gessert CE, Lowe L: Haemophilus influenzae type b in a day care center: Relationship of nasopfiaryngeal carriage to development of anticapuslar antibody. Pediatrics 65:65, 1980.
T. V. Murphy, M.D., Dan Granoff, M.D., D. F. Chrane, R.N., K. D. Olsen, B.S., S. J. Barenkamp, M.D., S. F. Dowell, and G. H. McCracken, Jr., M.D. Dallas, Texas, and St. Louis, Missouri
PHARYNGEAL COLONIZATION by H a e m o p h i l u s influenzae type b is present in fewer than 5% of healthy preschool children not exposed to a patient with systemic disease? ,2 By contrast, carrier rates as high as 50% have been found in preschool children who attend day care centers in which there was one or more individuals with Hib disease?' ~ Limited information is available describing the natural history of Hib colonization among children in day care in the absence of invasive disease. In 1963, Turk 5 reported that nine (11%) of 85 healthy preschool children in a day nursery without disease were colonized by Hib. More recently, surveys of children insix day care centers From the Department of Pediatrics, Southwestern Medical School, The University o f Texas Health Science Center, Dallas; and the Edward Mallinckrodt Department o f Pediatrics, Washington University School o f Medicine, and the Division o f Infectious Diseases, St. Louis Children's Hospital. Supported in part by the Zale Foundation and by Grant AI 17572 from the National Institute o f Allergy and Infectious Diseases. Submitted for publication July 9, 1984; accepted Sept. 21, 19841 Reprint requests: Trudy Murphy, M.D., Research Assistant Professor o f Pediatrics, The University o f Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235.
712
The Journal o f P E D I A T R I C S
where there was no H a e m o p h i l u s disease found only 3% (six of 223) colonization.6-8 We prospectively observed for 18 months well children who were enrolled in a day care center in Dallas and in whom there had been no known systemic H a e m o p h i l u s disease. Our objective was to determine Hib colonization in well children enrolled in day care who had no exposure to a patient with H a e m o p h i l u s disease. By using the strain Hib SDS-PAGE
Haemophilus influenzae type b
Sodium dodecyl sulfat~polyacrylamide gel eleetrophoresis
markers provided by biotyping and outer membrane protein gel analysis, we were able to identify specific strains colonizing children in the center.
METHODS The day care center studied was selected because, to the knowledge of the director, no child had been hospitalized with' invasive Hib disease in its 29-month history. The center was privately owned and operated for profit. Children came from families with a wide range of educational, economic, geographic, and ethnic backgrounds. Total
Volume 106 Number 5
enrollment during the study ranged from 80 to 100 children. Attendees were grouped by age. Infants (aged 1.5 to 17 months) were cared for by a single caretaker in a separate building. Toddlers (aged 18 to 35 months), a preschool group (36 to 71 months), and children who attended after school (5 to 8 years) were housed in different rooms of a second building and shared a common play yard. Children in the preschool group were further subdivided for learning activities approximately 2 hours each day. Meals were prepared by a cook and were eaten in the assigned classrooms. Children were promoted from one group to the next throughout the year after attaining the next age level. Infants had no contact with older groups except during the 2 weeks before promotion. At this time, an infant spent 2 hours a day with the toddlers. Study design. Parents of children at the day care center were asked to participate in the study by the center director. Informed, written consent was obtained for a single, monthly pharyngeal culture from each participant. The investigators made two visits to the center each month to obtain the cultures. N O change was made in the existing child care and educational programs. The study was conducted between December 1980 and May 1982. Absences related to illness were monitored by the center director and reported to the investigators. Illness consistent with invasive Hib disease or resulting in hospitalization of a child was investigated. We did not monitor minor illnesses or antibiotic usage. During March, 1981, 10 children with active rhinorrhea were observed during play time to determine possible modes of exchange of nasal secretions. Cultures of nasal secretions were obtained from the upper lip of three colonized children, on three occasions spaced by at least 1 day. These children also were asked to cough onto a chocolate agar plate placed an inch from the mouth. Pharyngeal cultures. Cultures were obtained by thorough swabbing of both tonsils and the posterior pharynx. Swabs were placed in brain-heart infusion broth supplemented with 5% Levinthal base and 5 U / m l bacitracin for overnight incubation at 37 ~ C in 5% CO2. Aliquots of overnight broth were plated onto chocolate and Hib antiserum agar, prepared as previously described.9 Plates were incubated in 5% CO2 at 37 ~ C for 24 hours and examined for colonies with typical morphology on chocolate agar or precipitin halos on antiserum agar. ~~ This method provided rapid screening of large numbers of samples and identification of Hib organisms present in low concentrations. H. influenzae was identified by growth requirements for X and V factor. H Capsular typing was confirmed by slide agglutination with commercial Hib antiserum (Difco Laboratories, Detroit). Isolates were assigned to one of six biotypes using the method of Kilian. H
a . influenzae type b infection in day care centers
71 3
The outer membrane protein patterns of Hib isolates were examined by SDS-PAGE using two gel systems. ~2 Subtype designation was assigned using the results of both gels) 3 Outer membrane protein subtypes were determined for H i b isolated from each child's first and last positive culture. In addition, all Hib isolates from four children who had multiple positive cultures were examined by these ~techniques. In each of these children consecutive isolates showed the same respective outer membrane protein subtype.. In the remaining children, if first and last isolates were identical by biotype and outer membrane protein subtype, respectively, then Hib isolated from consecutive intervening months were tested only for biotype. If concordant, these isolates were presumed to have the same outer membrane protein subtype a s the first and last isolates for calculation of the duration of colonization. For purposes of our study, two Hib organisms were considered to be the same strain if the respective biotypes and outer membrane protein subtypes were identical. The incidence of Hib colonization was defined as the number of children with at least one positive culture for Hib divided by the number of children participating in the study. The duration Of colonization was defined as the number of months of positive cultures for a given strain of Hib. When positive cultures were obtained both before and after one or two negative cultures, the negative cultures were counted as if positive for this calculation. Acquisition of Hib was defined as a positive culture after three negative cultures. Positive cultures during the first 3 months of the study were considered new acquisitions. RESULTS Culture specimens were obtained i n 83 children and 13 day care staff during the 18-month study. Data from 66 children and 12 staff who participated for >--5 months are presented. This subpopulation comprised between 40% and 50% of the center's total monthly enrollment, was similar in demographic features in the total population, and is referred to as the study population. Sixty-one percent of study children were white, 26% were black, 12% were Hispanic, and 2% were Asian. Fifty-three percent were boys. Eleven sets of siblings participated in the study, and three caretakers had a total of four of their own children enrolled in the center. Subjects remained enrolled in the study for a mean of 11.8 months (median 10 months) (Table I). Twenty-five children participated for the entire 18 months. Accretion and attrition rates of subjects participating and leaving the study averaged 1.8 and 1.2 subjects per rtrbnth, respectively. Thirty-two percent of study children were promoted to the next older age group while participating, including seven of 17 infants, 14 of 21 toddlers, and none of 28 pre-
7 14
Murphy et al.
25
The Journal of Pediatrics May 1985
84
zo = '15
40 e
s
z
T P T P T P T P T P T P Dec-Feb Mor-Moy Jun-Aug Sept-Nov Dec-Feb Mar-MQy '80 '8t '82
Figure. Quarterly prevalencerate of Haemophilus influenzae type b, subtype 1L pharyngeal colonization in children assigned to toddler and preschool groups. At least one pharyngeal culture was obtained from each child during calendar quarter. Absolute number of children assigned to toddler group decreased after third quarter of study, whereas preschool group increased in number. Hib subtype 1L was not isolated during last quarter of study. Table I. Number of months of study participation by group assignment
Group
Age (mo)
Number tested*
Months of follow-up (mean ++_SD)
Infant Toddler Preschool After-school Staff
1.5 to 17 ! 8 to 35 36 to 71 >72 Adult
17 28 35 7 12
6.6 • 315 8.8 • 4.6 8.9 • 5.3 12.7 • 6.0 12.8 _+ 3.0
*In this analysis, children promoted from one age group to another were counted in both groups.
Table II. Acquisition of pharyngeal colonization with Haemophilus influenzae type b by children and staff in a day care center
Group
Number cultured
Number positive
Incidence*
Infant Toddler Preschool After-school Staff
17 21 21 7 12
1t 15 10 1 1
6 71 48 14 8
*Number of children with at least one culture positive for Hib per 100 children in the center during 18 months of study, "~Hib colonization acquired after promotion to toddler group.
and after-school children. Fourteen of 17 infants enrolled after the study began. Hib colonization. During the 18 months, 750 cultures were obtained from children, and 154 from staff. Eightynine (10%) cultures were positive for Hib. The prevalence rate of colonization was 7% in the first month of the study,
and was greatest 4 months later (23% in March 1981). The rate was 0% during the last 2 months of the study. Seventy-one percent and 48% of children assigned to the toddler and preschool groups, respectively, had at least one positive culture for HIB during 18 months (Table II). Acquisition of Hib colonization was greatest during the first 6 months (December to May) of the study, averaging 2.2 children per month in the toddler group (13 of 17 children) and 1.2 children per month in the preschool group (seven of 16). During the last 12 months, the rate of acquisition of Hib was lower, averaging 0.25 child per month (six of 28) for children assigned to either group. Of the seven infants who were promoted to the toddler group during the study, only one became colonized by Hib. This infant was the only one transferred during the first 6 months of the study, when acquisition rates were highest. Colonization lasted for an average of 2.4 months per acquisition (median 1.0 month, range 1 to 7 months). The mean duration of colonization for toddlers was similar to that for children in the preschool group (2.6 a n d 2.3 months, respectively). Isolate characterization. The biotype of the 89 Hib strains isolated during the study fell into five of six groups described by Kilian. However, 93% of isolates were biotype 1. Eighty-four percent of acquisitions with biotype 1 organisms were associated with isolates of the outer membrane protein subtype 1L. The remaining isolates were either subtype 1H (13%) or 2L (3%). The prevalence of colonization by 1L organisms in the toddler and preschool groups, by calendar quarter, is shown in the Figure. This strain was the predominant isolate at the center for 15 of 18 months, and was the only strain that colonized children in all groups except the infants. It appeared to have been present initially in the toddlers, in which it became most prevalent, and then to have spread to the preschool children. Subtype 1H Hib was isolated from four children in the preschool group. One child was colonized for 3 months at the beginning of the study, and three others had positive cultures for 1 month only during March 1982. These three children previously had been carriers of 1L strains for durations of 2 to 6 months. Hib subtype 2L was isolated from a single child in the preschool group on three occasions over 8 months. Nasal secretions in toddlers. Cultures of nasal secretions from the upper lip of three toddlers who previously had been identified as Hib carriers werepositive for Hib seven of nine times over a 6-day period. Negative cultures resulted when nasal secretions had dried. Hib was not recovered from any of the "cough" plates held 1 inch from the mouth of five toddlers colonized by Hib. Ten children with rhinorrhea who were assigned to the
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toddler group were selected for observation of the handling of their nasal secretions. Total observation time was 150 minutes. Wiping or picking the nose, followed by placing the "contaminated" hand on a toy or other inanimate object occurred six times during this period. Coughing or sneezing occurred twice. Caretakers cleaned the rhinor 2 rhea with paper tissues three times but did not wash their own hands or the hands of the children. Nasal secretions appeared to be spread most efficiently at meals, an activity not included in the planned observations. Children shared food (mixed with nasal secretions) by taking a handful of food from their own mouth and placing it into the next child's mouth. On one occasion, an adult caretaker was observed to clean the faces of all toddlers with a single moist washrag. No invasive Hib disease was identified in study subjects or in members of their families during the 18 months. One child in the preschool group received treatment as an outpatient for pneumonia of undetermined cause during July 1981. Hib 1L had been isolated from the pharynx of this child in December 19801 but not during the intervening 7 months. Another infant who had left the day care center 2 months after the study began (and who therefore was not included in the study) developed osteomyelitis 9 months later from an Hib isolate with the 1H subtype. Hib had not been isolated from the pharynx of this child while she was at the center. DISCUSSION We have demonstrated that children in a day care center selected only because there were no proven cases of disease had high rates of acquisition and colonization by type b H. influenzae. High rates of colonization have been observed previously among children in day care, but nearly always in children exposed to a patient with type b Haemophilus disease. Recently, point prevalence studies at six centers without disease demonstrated relatively low rates of colonization by Hib (0% to 7%), 68 similar to those found in children who do notattend day care (1.4%). 2 HOwever, it is possible that higher rates would be found among children in day care if the children were observed for longer periods. At the Frank Porter Graham Development Center, where colonization has been monitored for more than a decade in the absence of disease, fewer than 5% of children were colonized by Hib for 87% of the months from 1970 to 1980. However, colonization was 5% to 10% for 11% of months, and between 10% and 20% for 1.6% of months during this period (Dr. Floyd Denny, University of North Carolina, Chapel Hill, personal communication, 1984). In our study, a single strain of Hib (1L) was found to colonize 67% of toddler and 33% of preschool group children, and to persist during 15 of the 18 months that
1t. influenzae type b infection in day care centers
715
cultures were obtained. The Hib subtype 1L strain was probably introduced into the center before the study began, possibly into the toddler group. Colonization of two children assigned to the toddler group (and the mother of one of them) was demonstrated in the first culture survey. Persistence of Hib colonization has been reported among contacts of individuals with disease in day care for np to 14 months. ~4 In our study center, without cases of invasive disease, Hib 1L was present in some children for 15 months. It is not known whether persistence of Hib among these children was related to characteristics of the particular Hib strain or to other factors (e.g., physical characteristics of the center, behavior patterns of the children, intercurrent viral infection). Three children who were carriers of Hib 1L were later Colonized by Hib 1H for a shorter period. However, it is possible that these children were colonized simultaneously by more than one strain. Dual coloniza~km would have gone unrecognized, because single colonies were selected for storage and characterization. Nevertheless, the second strain was detected only after one to three negative cultures, suggesting that colonization by Hib organisms with the 1H subtype represented new acquisitions. The lack of invasive disease among our study population is of particular interest, considering the high acquisition rates of potentially pathogenic Hib strains. When grown on solid media, all of the organisms were mucoid and formed strong precipitin halos on Hib antiserum agar? ~ Hib organisms with the 1L, 1H, or 2L subtypes have all been associated with invasive disease? z In a recent survey, 1L organisms accounted for only 7% of isolates from 256 patients hospitalized in 22 states~3; nevertheless, isolates with this subtype once may have been the predominant disease-producing strains in the United States. ~5 Furthermore, Hib organisms with the 1L subtype were isolated from four patients during a day care center outbreak of Hib disease within the same metropolitan area while our study was in progress. ~6 One possible reason for the lack of invasive disease in our study was the failure of Hib to colonize the youngest children, who would have been at greatest risk for developing disease, by virtue of their lack of immunity.17In the day care center studied, infants were isolated physically in their own building, and few infants were promoted to the toddler group during the early months of the study, when acquisition rates were greatest. In addition, the total number of infants studied was small. Colonized children at the center were recognized only because cultures were taken for the sttttty. There are limited data to suggest that healthy children with random cultures positive for Hib are not at increased risk for disease? For the present, prophylactic treatment with rifampin would not appear to be indicated for children who
7 16
Murphy et al.
are incidentally found to be H i b carriers, in the absence of exposure to a patient with Haemophilus disease. F r o m our results, it is suggested t h a t child day care provides a potential environment for high rates of acquisition of Hib, even in the absence of invasive disease. Acquisition of Hib colonization is associated with developm e n t of large concentrations of systemic antibody, 8.~7 conferring i m m u n i t y against disease. However, determining the actual risk of developing invasive disease r a t h e r t h a n i m m u n i t y will require a larger, prospective study. F u r t h e r research is necessary to define those f a c t o r s - microbial, host, or e n v i r o n m e n t a l - - t h a t m a y contribute to development of the invasive disease in a given population. We thank Mrs. Elaine Krause, director of the study day care center, for allowing the study to take place and for obtaining informed consent from parents of enrolled children; Mrs. Teresa Zweighaft for laboratory assistance during the first 6 months of the project; Marian Troup for preparation of the manuscript; and Katherine McCullough for technical assistance. J. B. Robbins, M.D., Bureau of Biologics, Bethesda, Maryland, provided hyperimmune burro anti-Hib serum. REFERENCES
1. Michaels RH, Poziviak CS, Stonebraker FE, Norden CW: Factors affecting pharyngeal Haemophilus influenzae type b colonization rates in children. J Clin Microbiol 4:413, 1976. 2. Hampton CM, Barenkamp S J, Granoff DM: Comparison of outer membrane protein subtypes of Haemophilus influenzae type b isolates from healthy children in the general population and from diseased patients. J Clin Microbiol 18:596, 1983. 3. Granoff DM, Daum RS: Spread of Haemophilus influenzae type b: Recent epidemiologic and therapeutic considerations. J PEDIATR 97:854, 1980. 4. Barenkamp S J, Granoff DM, Munson RS Jr: Outermembrane protein subtypes of Haemophilus influenzae type b and spread of disease in day care centers. J Infect Dis 144:210, 1981. 5. Turk DC: Naso-pharyngeal carriage ofHaemophilus influenzae type b. J Hyg Camb 61;247 , 1963.
The Journal of Pediatrics May 1985 6. Ward JL, Gorman G, Phillips C, Fraser DW: Haemophilus influenzae type b disease in a day care center: Report of an outbreak. J PEDIATR 92:713, 1978. 7. Gran0ff DM, Gilsdorf J, Gessert C, Basden M: Haemophilus influenzae type b disease in a day care center: Eradication of carrier state by rifampin. Pediatrics 63:397, 1979. 8. Cox F, Trincher R, Rissing JP, et al: Rifampin prophylaxis for contacts of Haemophilus influenzae type b disease. JAMA 245:1043, 1981. 9. Murphy TV, Chrane DF, McCracken GH Jr, Nelson JD: Rifampin prophylaxis versus placebo for household contacts of children with Haemophilus influenzae type b disease. Am J Dis Child 137:627, 1983. 10. Michaels RH, Stonebraker FE, Robbins JB: Use of antiserum agar for detection of Haemophilus influenzae type b in the pharynx. Pediatr Res 9:513, 1975~ 11. Kilian M: Haemophilus. In Lanette EH, editor: Manual of clinical microbiology, ed 2. Washington, D.C., 1980, American Society for Microbiology, pp 330-336. 12. Barenkamp SJ, Munson RS Jr, Granoff DM: Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. J Infect Dis 143:668, 1981. 13. Granoff DM, Barenkamp SJ, Munson RS Jr: Outer membrane protein subtypes for epidemiologic investigation of Haemophilus influenzae type b disease. In Sell SH, Wright PF, editors: The biology of Haemophilus influenzae. New York, 1982, Elsevier Science Publishing Co., pp 43-55. 14. Ginsburg CM, McCracken GH Jr, Rae S, Parke JC Jr: Haemophilus iJ~uenzae type b disease: Incidence in a day care center. JAMA 238:604, 1977. 15. Barenkamp S J, Granoff DM, Pittman M: Outer membrane protein subtypes and biotypes of Haemophilus influenzae type b: Relation between strains isolated in 1934-1954 and 1977-1980. J Infect Dis 148:1127, 1983. 16. Murphy TV, McCracken GH Jr, Moore BS, et al: Haemophilus influenzae type b disease after rifampin prophylaxis in a day care center: Possible reasons for its failure. Pediatr Infect Dis 2:193, 1983. 17. Granoff DM, Gilsdorf J, Gessert CE, Lowe L: Haemophilus influenzae type b in a day care center: Relationship of nasopfiaryngeal carriage to development of anticapuslar antibody. Pediatrics 65:65, 1980.