Outbreaks of human enteric adenovirus types 40 and 41 in Houston day care centers

Outbreaks of human enteric adenovirus types 40 and 41 in Houston day care centers Rory V a n , DrPH, C h u a n - C h u a n Wun, MS, M i g u e l L. O'Ryan, MD, David O. M a t s o n , MD, PhD, Laurie Jackson, BS, a n d Larry K. Pickering, MD From the Division of Infectious Diseases, Department of Pediatrics, University of Texas Medical School at Houston, and the Department of General Medicine, Department of Pediatrics and Division of Molecular Virology, Baylor College of Medicine, Houston, Texas

Objective. Human enteric adenovirus (EAd) types 40 and 44 cause diarrhea in y o u n g children, but little is known about their association with outbreaks of diarrhea in the child care setting. This study e v a l u a t e d EAd as a cause of outbreaks of diarrhea a m o n g infants and toddlers in d a y care centers. Design. Stool specimens were c o l l e c t e d w e e k l y regardless of symptoms during four periods from January 1986 to April 1991, from children 6 fo 24 months of a g e enrolled in prospective studies of diarrhea in d a y care centers. All diarrhea stool specimens were tested for bacterial enteropathogens, rotavirus, and Giardia lamblia. A total of 431 outbreaks occurred during the study. No e t i o l o g i c a g e n t was identified in 77 outbreaks. Stool specimens from 75 of these 77 outbreaks and from another 21 outbreaks of diarrhea with a known cause were e v a l u a t e d for EAd with a m o n o c l o n a l a n t i b o d y - b a s e d enzyme immunoassay. Results. A total of 4402 stool specimens from 613 children from these 96 outbreaks was tested for EAd. The virus was d e t e c t e d in specimens c o l l e c t e d during 10 outbreaks, 3 of which occurred in 1986, 3 in 4988, 4 in 4989, 4 in 1990, and 2 in 4991. Of 249 children, 94 (38%) in these 10 EAd outbreaks were infected with EAd. In 51 children (54%) the infection was symptomatic and in 43 (46%) it was asymptomatic. Outbreaks lasted 7 to 44 days (mean 24.5 days). Duration of EAd excretion ranged from I to 14 days (mean 3.9 days), with excretion occurring from 7 days (mean 2.6) before diarrhea b e g a n to 11 days (mean 5.3 days) after diarrhea stopped. Conclusion. Enteric adenovirus types 40 and 44 are an important cause of outbreaks of diarrhea a m o n g children attending d a y care centers, often involve children in more than one room, and frequently p r o d u c e asymptomatic infection. (J PEDIATR1992;120:516-24)

Although most adenoviruses cause respiratory tract diseases, a few are associated with gastroenteritis. Forty-two different serotypes of human adenoviruses are currently Supported in part by National Institutes of Health grant No. HD 13021. Presented in part at the annual meeting of the Society for Pediatric Research, New Orleans, La., May 1991. Submitted for publication Sept. 24, 199i; accepted Nov. 14, 1991. Reprint requests: Larry K. Picketing, MD, University of Texas Medical School, 6431 Fannin St., JFB 1.739, Houston, TX 77030. 9/20/35055 516

well defined, and an additional five serotypes have been proposedJ All serotypes are classified into one of six biologic groups (A to F) on the basis of their genornic properties. Enteric adenovirus types 40 and 41 belong to group EAd EIA

Enteric adenovirus(es) Enzyme immunoassay

[

I

F and are the two serotypes consistently associated with diarrheal illness in young children.2, 3 Studies in both community and hospital settings in various countries have demonstrated that EAd have a worldwide distribution and that

Volume 120 Number 4, Part 1

Enteric adenovirus types 40 and 41 in day care centers

5 17

T a b l e I. Study periods and number of participating day care centers and children Monitoring periods (No. of months)

No. of centers*

No. of children

Child-months of surveillance

1/86 to 3/87 (15) 13t 635 12/87 to 4/88 (5) 7 226 1/89 to 3/89 (3) 6 94 10/89 to 4/91 (19) 4 355 *Sevenday care centersparticipatedin morethan one studyperiod. tOne day care centerclosedin September 1986.A replacementcenter was enrolledin October 1986.

by the age of 4 years, more than 50% of children have serum antibodies,4, 5 indicating previous infection. Studies of diarrhea in infants and toddlers in day care centers have established that the incidence of diarrhea is high and that most episodes occur during outbreaks. 6 Although diagnostic testing for many enteropathogens has been performed on stool specimens from children involved in outbreaks, the etiologic agent of approximately 50% of these outbreaks remains undefined. The hypothesis of this study was that EAd was responsible for some day care center diarrhea outbreaks of unknown cause. For this study, we identified outbreaks of diarrhea associated with EAd involving Children in day care centers; determined the ratio of symptomatic to asymptomatic EAd infections during the outbreaks; and described the duration of EAd outbreaks and the pattern of Virus excretion by children during outbreaks. METHODS Study design and population. This study was part of a prospective, longitudinal surveillance of diarrheal disease in children younger than 24 months of age who were enrolled in 17 day care centers in Houston, Tex. The four study periods were as follows: January 1986 to March 1987, December 1987 to April 1988, January t OMarch 1989, and October 1989 to April ! 991 (Table I). The number of monitoring periods and the individual centers participating in each period changed. In the first monitoring period, 13 centers participated; in thesubsequent monitoring periods, seven, six, and four centers, respectively, participated. The number of children monitored during each period ranged from 94 to 635, for a total of 6908 child study-months of surveillance. All centers that participated in the study were licensed by the Texas Department of Human Services and were located within a 5-mile radius of the University of Texas Medical School at Houston. Permission was obtained from the day care directors and care givers and from the parents of each child studied. Questionnaires requesting demographic data were administered and completed at enrollment. This study was approved by the committee for the protection of human

3888 645 225 2150

No. of diarrhea outbreaks

60 16 4 51

subjects of the University of Texas Health Science Center at Houston. Diarrhea surveillance. Infants and toddlers enrolled in the study were monitored for diarrhea by the care givers. When there was no diarrhea in the center, each study day care center was visited by a research nurse twice weekly and contacted by telephone daily. Once diarrhea, fever, or vomiting was identified in a study child in a center, a research nurse visited the center daily to obtain detailed information regarding the illness. When a child was absent because of illness, information on whether diarrhea had occurred during the absence was obtained from the parent directly or through the center's care giver. Each enrolled child was monitored until the study period ended or until the child moved to a nonstudy room or left the center. During each study period, children newly attending the day care centers and --<24 months of age were enrolled. None of the cleaning, diapering, or housekeeping procedures were altered during this study. Definitions. Diarrhea was defined as the passage of unformed stools with at least twice the usual daily frequency. A diarrhea episode was defined as the occurrence of diarrhea, separated from previous diarrhea in that child by a period of more than 7 days without symptoms. Children were placed into day care center rooms by age according to the policy of each day care center. A total of 71 day care center rooms were monitored (range two to four rooms per day care center). An outbreak of diarrhea was defined as the occurrence of diarrhea in two or more children in a day care center room within 1 week. Outbreaks in the same day care room were required to be separated by 7 consecutive days during which all children in the day care center room were free of diarrhea. An E A d infection was defined as the detection of EAd in a stool specimen. An E A d outbreak was defined as the occurrence of EAd in stool specimens from two or more children regardless of symptoms in a day care center room during a study week. Stool collection. All children enrolled in the study wore disposable diapers while at the day care center. Stool specimens were collected routinely from each child on a weekly basis. Whenever diarrhea was identified, stool specimens

5 18

Van et al

were collected twice weekly from children having diarrhea. These specimens were collected at the time of diaper changes by day care center care givers or by study nurses. Specimens were divided into aliquot portions and placed in appropriate transport media tubes and containers, and brought in refrigerated, temperature-controlled containers to the research laboratories for testing. Remaining raw stools were stored at - 7 0 ~ C. Identification of enteropathogens. Each child in whom diarrhea developed during the study period had a stool specimen tested for Shigella, Salmonella, Campylobacter jejuni, Aeromonas, Yersinia enterocolitica, Plesiomonas shigelloides, and Escherichia coli, ser0type O157:H7, by means of standard laboratory microbiologic procedures] Enzyme immunoassay methods were used to detect EAd, group A rotavirus, and Giardia lamblia antigens, s, 9 Rotavirus testing was performed on all stool specimens collected, regardless of symptoms. Testing of EAd with the Adenoclone 40/41 EIA (Cambridge Biotech Corp., Worcester, Mass.) was performed on all diarrhea specimens. This assay has been shown to have a sensitivity and specificity of 98%, in comparison with electron microscopy and D N A restriction enzyme analysis. For study period 1, only outbreaks without a known cause were evaluated for the presence of EAd. For study periods 2 to 4, all outbreaks of diarrhea were tested for EAd. Statistical analysis, Stool collection, demographic data, and EAd testing results were entered into an IBM-compatible microcomputer and edited with a Paradox 3.01 data management software package (Borland International, Inc., Scotts Valley, Calif.). Results of EAd testing and diarrhea outbreak data were analyzed by SAS-PC (6.04 release) (SAS Institute, Inc., Cary, N.C.). The percentage of EAd infection was calculated on the basis of the number of children infected with EAd and the number of children tested during each outbreak period in the day care centers. Logistic regression analysis was used for statistical testing of the association of EAd infections with diarrhea, the relationship between the age of children (in months) and the occurrence of EAd infections, and the effect of individual day care center rooms on the occurrence of EAd infection. The Student t test was used to compare the means of age and the duration of EArl excretion between children with symptomatic infections and those with asymptomatic infections. RESULTS Study sites and population. Seventeen day care centers participated in this longitudinal study. Seven centers participated during more than one study period. A total of 1310 children were enrolled and monitored for the occurrence of diarrhea.

The Journal of Pediatrics April 1992

Enteropathogens associated with outbreaks of diarrhea. During the 309 day care center study-months there were 131 outbreaks of diarrhea, which included 1207 episodes of diarrhea. The number of children involved in each outbreak ranged from 2 to 55, with a mean of 9. At least one enteropathogen other than EAd was identified in association with 54 (41%) of the 131 diarrhea outbreaks. Thirty-three outbreaks (25%) were associated with rotavirus alone, 8 (6%) with G. lamblia alone, 8 (6%) with rotavirus and G: lamblia, 2 (1.5%) with Aeromonas spp. and G. lamblia, 1 (1.0%) with Shigella sonnei and G. lamblia, and 1 each with calicivirusl~ and Aeromonas spp. alone. Detection of enteric adenovirus and symptom status. A total of 4402 stool specimens from 613 children were tested for E A d types 40 and 41, including 967 specimens from children with diarrhea and 3435 specimens from children without diarrhea. Of the 77 outbreaks of diarrhea with no known cause, 25 of the 27 from study period 1 were further evaluated for EAd, which was identified in 3. During study periods 2 to 4, all 71 outbreaks of diarrhea (50 without a known cause) were tested for EAd, which was identified in 7 (3 in period 2 and 4 in period 4) (Table II). Although calendar months were not sampled equally (Table I), we noted outbreaks in every calendar month of the year except May, June, July, and November. During the EAd outbreaks, 6 outbreaks had 2 rooms involved in the day care centers, 2 had 3 rooms involved, and one each had 1 and 4 rooms involved. There were 249 children from nine different day care centers involved in the 10 EAd outbreaks; EAd infection was documented in 94 children (38%). Infection in each outbreak ranged from 20% to 60% (mean 38%). Fifty-one children (54%) were symptomatically infected and 43 (46%) had no symptoms during infection (Table II). To evaluate the sensitivity and specificity of the Adenoclone 40/41 EIA, we sent 417 stool specimens to the Centers for Disease Control Viral Gastroenteritis Unit, where they were tested in an EIA that incorporates reagents developed by the Centers for Disease Control. The sensitivity and specificity of the two EIAs were 95% and 100%, respectively. We are currently performing D N A restriction enzyme analysis on stool specimens with EAd. Duration of EAd excretion. The mean duration of the EAd outbreaks was 24.5 days (range 7 to 44 days). The mean duration of EAd excretion by children was 3.9 days (range 1 to 14 days) (Table II). Nine children were excreting EAd before diarrhea occurred (mean 2.6 days, range 1 to 7 days). Ten additional children continued to excrete EAd after their diarrhea had Stopped for a mean of 5.3 days (range 1 to 11 days). Four children (other than the 19 described above) were intermittent EAd excreter s: they had formed stools without detectable EAd in between watery

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Enteric adenovirus types 40 and 41 in day care centers

5 19

Table II. N u m b e r of children infected, symptom status, and duration of enteric adenovirus outbreaks

Outbreak No.

l 2 3 4 5 6 7 8 9 10

DCC

Outbreak period

A* A* B C D E F G H I

Feb-Mar 1986 Aug 1986 Sept-Oct 1986 Jan1988 Feb 1988 Jan 1988 Dec 1989 Sept 1990 Mar-Apr1991 Mar-Apr 1991

Children infected with EAd

No. of children exposed

Exposed children infected with EAd No.

%

No.

%

No,

%

13 27 20 28 27 17 25 31 19 42 249t

7 10 12 10 7 4 5 9 11 19 94t

54 37 60 36 26 24 20 29 58 45 38~

4 6 3 6 2 3 1 7 4 15 51t

57 60 25 60 29 75 20 78 36 79 54~

3 4 9 4 5 1 4 2 7 4 43t

43 40 75 40 71 25 80 22 64 21 46~

With diarrhea

Well

Outbreak duration (days)

44 23 41 22 15 19 7 18 17 39 24~

Mean duration of excretion per infected child (days)

6.4 5.4 4.3 4,6 2.4 5.0 2.0 3.3 2.3 3.4 3.9~

DCC, Day care center. *Eight children were exposed to an Ead during both outbreaks in day care center A; one child was infected twice, "~Totalnumber of children. :~Mean of values listed in column.

stools in which EAd was detected. The virus was excreted for longer periods by the children with symptomatic infections than by the children whose EAd infections were asymptomatic (4.2 _+ 0.4 days vs 2.8 + 0,5 days, respectively; p = 0.04). Association between EAd and other enteropathogens. Of the 10 E A d outbreaks identified during the entire study period, 6 were associated with EAd alone. In 3 of these outbreaks only E A d was identified; in the other 3, additional enteropathogens were identified only in well children. In 4 outbreaks, other enteropathogens were identified. In 3 outbreaks, 4 children were coinfected with E A d and another enteropathogen on the same date. Other children in outbreak rooms were infected with other enteropathogens, but not with EAd, during the outbreak period. These children were infected with G. lamblia, rotavirus, or Aeromonas. In day care center A, two separate E A d outbreaks were identified in 1986, one in February and March and the other in August. Eight children were exposed to both EAd outbreaks. One of the eight children was asymptomatically infected with EAd both times, and two others were infected only during the first EAd outbreak (one with symptomatic and one w i t h a s y m p t o m a t i c infection). Relationship between the age of the children and EAd infections. As the child's age increased, the likelihood of being infected by E A d decreased significantly (p = 0.02). The mean age of EAd-infected children (13 months) was significantly younger than that of children uninfected with E A d (15 months) (p = 0.02). Children with symptomatic and those with asymptomatic infections were of similar age (mean 12 and 13 months, respectively).

EAd infection and day care center room. Infections with E A d were not found to be equally distributed among the day care centers studied. In 8 of 17 centers, no E A d was detected. Even within centers where EAd was d e t e c t e d , E A d activity varied in intensity among rooms, The 9 day care centers in which EAd were detected contained 25 rooms. In 2 of t h e 2 5 rooms the E A d infections ~were not detected during the monitoring periods, in 5 rooms 1 infected child was identified, and in the remaining 18 rooms 2 or more infected children were identified (p <0.001, multiple logistic regression). We also evaluated whether the presence and number of EAd infections, the day care room, and the presence or absence of rotavirus infection were independent factors, each increasing the likelihood of an outbreak of diarrhea in a room. The effect of the particular day care room on the occurrence of diarrhea was evaluated either without adjustment or after adjustment by the presence of EAd infection, by the presence of rotavirus infection, or by the concurrent presence of both EAd infection and rotavirus infection. In each of the four analyses the day care center room remained an independent determinant of the presence of an outbreak of diarrhea (Wald chi-square analysis, d f = 25, p <0.001). DISCUSSION Enteric adenovirus types 40 and 41 have been reported to cause gastroenteritis in children in hospitals, outpatient clinics, and communities. 1117 One outbreak of infection with EAd types 40 and 41 has been reported in each of a long-stay children's ward, a camp in the United Kingdom, and an orphanage in Japan. 18-2~Sporadic illness affecting

52 0

Van et al

The Journal of Pediatrics April 1992

48%

Unknown

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H

Ih]]lllll]llll

~'~--.~.l:l::I:;;:::::::lll::/ll~Ilql~ Shlgnlla !

1%

~ - - - ~ - - ~ Aeromona~ and Giardta ~

Rotavirus

and Gtardla

2

2%

Caliclvirus3%

Rotavtrus

an~

Gtarata

6%

)

Glar~la 6% Enteric

aaenovirose,

s

8%

Figure. Enteropathogens associated with outbreaks of diarrhea in Houston day care centers. Calicivirus percentage was based on testing of 36 outbreaks.

children in day Care centers in Copenhagen and Arizona21, 22 has been reported, but EAd outbreaks in day care centers have not been reported. In this study, a large number of specimens from 613 children involved in 96 outbreaks of diarrhea in four sequential study periods were analyzed. Ten EAd outbreaks were detected, making EAd second only to rotavirus as a cause of outbreaks of diarrhea in this setting. Despite testing for 10 enteropathogens, 63 (48%) of the 131 outbreaks from our total study period do not yet have a known cause (Figure). These outbreaks are currently under evaluation for other potential viral enteropathogens. The development and use of diagnostic tests for the detection of other agents will reduce the percentage of outbreaks of unknown cause. The longitudinal design of this study, in which stool specimens were collected weekly from each child, enabled us to identify EAd infection regardless of symptoms of diarrhea. The finding that 46% of infected children were symptom free is similar to that reported for rotavirus infections in children in day care centers23 and may account for the fact that by 4 years of age, the majority of children are seropositive for EAd. The mean duration of EAd excretion by children with symptomatic infection was 4.2 days, and by children with asymptomatic infection 2.8 days. Actual excretion days were likely to have been longer, but the weekly stool specimen collection procedure precluded a more specific determination. The method of specimen collection in this study did permit us to detect EAd excretion by children as early as 7 days before diarrhea began and as late as 11 days after

diarrhea stopped. This excretion pattern is similar to that described for rotavirus. 23 The identification of multiple enteropathogens during outbreaks of diarrhea in day care center settings is common.6, 24, 25 In 4 of the 10 EAd outbreaks that we identified, rotavirus, G. lamblia, and Aeromonas were detected in stool from children with diarrhea who were not infected with EAd. Only four children were simultaneously infected with EAd and another enteropathogen. The occurrence of multiple enteropathogens during outbreaks indicates the difficulty one faces in determining the cause of individual outbreaks of diarrhea in child care center settings and the potential for the transmission of multiple enteropathogens simultaneously. EAd as well as rotavirus and Giardia spread not only among children within single day care center rooms but also to children in different rooms. Transmission between rooms may occur when toddlers wander from one room to another or during interactions in the common play areas, where young children from many rooms play with each other before and after spending time in their own rooms. Our study demonstrates that EAd is the second most common cause of outbreaks of diarrhea in the day care center setting. With the availability of EIA for detection of EAd, rotavirus, and astrovirus, 8, 22 and with the recent cloning of the Norwalk virus,26 it should be possible in the near future to establish a rapid viral diagnostic panel that will permit diagnosis of infection with these four enteric viruses. If one adds calicivirus to this panel, 1~then the spectrum of viruses that commonly cause gastroenteritis may be

I'D/'"III' 120 4, Pari I

Ell/eric UclC'II01'irus typc'.r % Clllcl of / {II cluy caf(' ('c'/IIc'rs

521

NUlllba

thoroughly evaluated. The epidemiologic information based on the application of thc newly availnble diagnostic assays will provide the clinician with knowledge of the cause of diarrhea and permit modification of costly evaluation lind intcrvention strategies. In late 1990 the Centers for Disease Control assembled a national surveillance system for reporting EAd infections. Participants from \'arious hospitals and laboratories nationwide are submitting periodic reports to the Centers for Disease Contro\. The data gathered frol11 the nationwide survey will permit n better understanding of the seasonal nnd regional distribution of EAd as n cause of gastroenteritis. Development of a vaccine ngainst EAd is premature at this time, bccause further information is neecssary about the molecular virologic and immunologic characteristics of this ngen!. Howcver, the frequency of infection with this agent indicates that a vaccine may be needcd. We thank Dr. Roger Glass and his group, in the viral gastroenteritis unit at the Centers for Disease Control; Dr. f\lary Estes, in the Dh'ision of Molecular Virology. Daylor College of Medicine, for technical support and ndvice; and Ardythe Morrow, PhD, Alison Hardy, DS, and Wcndy Malley for nssistance wilh data managemcnt. Wc also apprecinte the help of Melissa Carhart, RN, Kathy Franco, RN, Michelle Miller, RN, Irenc Townsend, RN, nnd nil the day care center children, parents, and care givers. REFERENCES I. Heirholzer JC, Wigand R, Anderson LJ, et al. Adenm'iruscs from patients with AIDS: n plethora of serotypes nnd a description of fh'e new serotypes of subgenus D (types 43-47). J Infect Dis 1988;158:804-13. 2. Horwitz MS. Adenoviruscs. In: Fields UN, Knipe DM, cds. Virology. New York: Ravcn Press, 1990. 3. Svensson L, Uhnoo I, Wndell G. Enteric ndenoviruses of man. In: Saif LJ, Theil KW, cds. Viral diarrhea of man and animals. Boca Raton, Florida: CRC Press, 1990. 4. Kidd AI-I, Danatvnla JE, dcJong JC. Antibodies to fnstidious faecal adenoviruses (species 40 and 41) in sera from children. J Med Virol 1983;11 :333-41. 5. Shinozaki T, Araki K, Ushijima 1-1, et a1. Antibody response to enteric adenovirus lypes 40 and 41 in scm from people in vnrious age groups. J Clin Microbiol 1987;25:1679-82. 6. Pickering LK, Evans DG, DuPont HL, et a1. Diarrhea caused by Shigella, rotaviruses, and Giardia in day C.1Te centers: prospccth'c study. J PI!DIATR 1981:99:51-6. 7. Balows A, Hausler WJ Jr, Herrmann KL, Isenberg liD, Shadomy I-IJ, cds. Manual of clinic.11 microbiology. 5th cd. Washington, DC: American Society for Microbiology, 1991. 8. Knisley C\', Dednarz-Pmshad AJ, Pickering LK. Detection of

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