Recovery of Giardia lamblia Cysts From Chairs and Tables in Child Day-Care Centers

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1006 SUPPLEMENT

lower. Determining the level of compliance is, therefore, essential to the evaluation of any public health intervention.

Investigation of multiple outbreaks attributable to the same agent in these settings has led to increased knowledge through applied epidemiologic principles.9’2”3 For example, Tauxe et al investigated outbreaks of shigellosis in two neighboring day-care centers, applied different interventions, and compared out-come5.’2 Outbreaks were controlled in both centers, although the

need for alternate care was 100-fold less among children attending

the center in which convalescent children taking antibiotics were cared for in isolation than among children who attended the center that temporarily closed.’2

CONCLUSION

The importance of disease surveillance and outbreak-control activities in child day-care settings has been detailed as part of the American Public Health Association,’American Academy of Pedi-atrics guidelines for out-of-home day-care programs.’4 Aggressive assessment of outbreaks will continue to provide critical informa-tion needed to prevent and control diseases and other adverse health events in day-care facilities.

REFERENCES

1. Thacker SP, Addiss 0G. Goodman RA, Holloway BR, Spencer HC. Infectious diseases and injuries in child day-care: opportunities for healthier children. JAMA. 1992;268:1720-1726

2. Klein JO. Infectious diseases and day care. Rev Infect Dis. 1986;8:521-526 3. Davis JP, Pfeiffer JA. Surveillance of communicable diseases in child

day care setting. Rev Infect Dis. 1986;8:613-617

4. Bartlett AV, Jarvis BA, Ross V. Diarrheal illness among infants and toddlers in day care centers: effects of active surveillance and staff

training without subsequent monitoring. Am I Epidemiol. 1988;127:

808-817

5. Pickering LK, Evans DG, DuPont HL, Vollet JJ, Evans DJ. Diarrhea

caused by Shigella, rotavirus, and Giardia in day-care centers: a pro-spective study. J Pediafr. 1981;99:51-56

6. Van R, Wun CC, O’Ryan ML, Matson DO, Jackson L, Pickering LK. Outbreaks of human enteric adenovirus types 40 and 41 in Houston day care centers. J Pediatr. 1992;120:516-521

7. Centers for Disease Control. Case definitions for public health surveil-lance. MMWR. 1990;39(No. RR-13):1-43

8. Lew JF, Moe CL, Monroe 55, et al. Astrovirus and adenovirus associated with diarrhea in children in day care settings. J Infect Dis. 1991;164: 673-678

9. Bartlett AV, Englender SJ, Jarvis BA, Ludwig L, Carlson JF, Topping JP. Controlled trial of Giardia lamblia: Control strategies in day care cen-ters. Am J Public Health. 1991;81:1001-1006

10. Dean AD, Dean JA, Burton JH, Dicker RC. Epi Info, Version 5: A word processing, database, and statistics program for epidemiology on mi-crocomputers. Atlanta: Centers for Disease Control; 1990

I I. Steketee RW, Reid 5, Cheng T, Stoebig JS, Harrington RG, Davis JP.

Recurrent outbreaks of giardiasis in a child day care center. Am IPublic Health. 1989;79:485-490

12. Tauxe RV, Johnson KE, Boase JC, Helgerson SD, Blake PA. Control of day care shigellosis: a trial of convalescent day care in isolation. Am

Public Health. 1986;76:627-630

13. Tacket CO. Cohen ML. Shigellosis in day care centers: use of plasmid analysis to assess control measures. Pediatr Infect Dis J. 1983;2:127-130 14. American Public Health Association/American Academy of Pediatrics.

Caring for our children. National health and safety performance standards: Guidelines for out-of-home child care programs. Washing-ton and Elk Grove Village IL: APHA/AAP; 1992

Recovery

of

Giardia

lamblia

Cysts

From

Chairs

and

Tables

in

Child

Day-Care

Centers

Mildred

M.

Cody,

PhD,

RD;

Henry

M.

Sottnek,

PhD,

MT(ASCP),

SM(NRM);

and

Virginia

S. O’Leary,

PhD

More than I I million children attend commercial child day care in the US. This number is expected to increase as more mothers of

preschool children enter the work force. Infectious diseases are

readily transmitted in child day-care settings,’4 where children

are in close contact with one another for approximately 50 hours

per week. Diarrheal diseases are common in these settings. Infec-tious diarrhea can be transmitted by person-to-person contact” and, possibly, by contact with fomites.’’ Many food service

sur-faces including tables, kitchen counters, ware-washing sinks, and dinner plates in the centers are contaminated with bacterial levels that exceed public health standards.6’7

Giardia lamblia is a pathogenic intestinal protozoan that may produce an acute infection characterized by diarrhea and other clinical symptoms.8 It infects children in day care at a higher rate than the general population.2’ Family members are at risk of acquiring C. lamblia transmitted from children attending

child-care centers3 where infectious cysts of G. lamblia may be shed by infected symptomatic or asymptomatic individuals.’ Intervention strategies that rely on exclusion of infected children from day-care settings are expensive and do not control Giardia infections in the child day-care environment.4

We compared the use of a commercially available, indirect

immunofluorescent procedure with direct microscopic examina-lion to detect G. lamblia cysts in stools of children attending day-care centers. Because the detection of G. lamblia on environ-mental surfaces would show their potential for transmitting

giar-From the College of Health Sciences, School of Allied Health Professions, Departments of Nutrition and Dietetics and Medical Technology, Georgia State University, Atlanta, GA.

Use of trade names is for identification only and does not constitute en-dorsement by the Public Health Service, the Centers for Disease Control and Prevention, or any of the other co-sponsors of this Conference.

diasis, we used the immunofluorescent procedure to look for G. lamblia cysts on various surfaces. Cysts were removed by swab-bing surfaces, recovered on membrane filters, and detected using the indirect immunofluorescent procedure on the filters. Then the procedure was field tested in six commercial child day-care centers.

METHODS

Giardiasis Survey of Field Sites

Subjects

Children 2 to 3 years of age and their care givers in six com-mercial day-care centers in Atlanta, GA, were tested for giardiasis. The centers, all part of the same national chain, were comparable in design and clientele. Center directors described the study in a

letter to parents of potential participants. Investigators answered

parents’ questions and explained the human subjects consent form required of all participants. After testing an original group of 80 children and care givers, family members of children who tested positive for G. lamblia and six additional children from the same centers were tested. A physician provided free treatment of in-fected individuals, when requested.

Stool

Collection,

Processing,

and

Examination

Kits included three specimen vials containing sodium acetate-acetic acid formalin (SAF) fixative-preservative (PARA-PAK SAF#{174};Meridian Diagnostics Inc. Cincinnati, OH) and directions for use. These kits were given to participants’ parents and to participating care givers. Parents collected three separate, consec-utive stool specimens from their children at home. Participating

care givers provided their own three separate, consecutive speci-mens. Investigators pooled half of each of the three specimens and

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SUPPLEMENT 1007 processed them by the PARA-PAK MACRO-CON#{174} modification

of the Ritchie formalin-ether concentration procedure (Meridian Diagnostics mc, Cincinnati, OH), substituting ethyl acetate for

ethyl ether. Students were trained to examine processed specimens both by light microscopy and by fluorescent microscopy with

Me-ridian Diagnostics’ MERIFLUOR-Gzard#{224}i indirect immunofluores-cent procedure. All specimens were examined at 100 x, confirmed at

400 x, and repeated by an experienced investigator.

Testing of Environmental Surfaces

Laboratory

Development

of Method

Cyst inoculum standards were prepared by pooling two SAF-preserved, Giardia-positive stool specimens and diluting to I :10, 1:50, 1:100, and 1:200 with 5% buffered formalin. These dilutions

were used to find the limits of cyst detection. To verify cyst

distribution, investigators counted each dilution by light micros-copy in a Neubauer hemocytometer.

Four different test surfaces-wood, coarse-textured fiberglass chairs, stainless steel, and Formica#{174}were experimentally

con-taminated with 10 pL of standard. Standards were placed on any

part of the test surface, spread with the plastic disposable tip of a micropipet, and allowed to air dry. Test surfaces measured 50 cm2 (5 cm X 10 cm) or 400 cm2 (20 cm X 20 cm). Thirty of the potential combinations were run (Table 1).

Samples were collected in vials containing 2 mL of

phosphate-buffered saline (PBS) and 20 pL of a surfactant (Brij-35#{174};Technicon Instruments Corp. Tarrytown, NY) (PBS-B), because 27 pilot trials without surfactant did not recover cysts from any test surface. Investigators placed I mL of PBS-B on each experimentally con-taminated test surface and swabbed each twice with two dry

cotton swabs. Swabs were returned to the remaining PBS-B, agi-tated vigorously, pressed against the side of the vial to remove moisture, and then discarded.

Samples were placed into 5-mL lure-lock syringes attached to

filter holders (Gelman Sciences, Ann Arbor, MI). Cysts were

con-centrated by force-filtering through Nuclepore#{174} polycarbonate

membranes (Nuclepore Corporation, Pleasanton, CA) supported

by the filter holders. The same MEfflFLUOR-Giardia indirect immunofluorescence procedure employed for stool examination

was used for on-filter cyst detection.

Field

Testing

of Method

Fifty-three coarse-textured fiberglass chairs and 53 Formica#{174}

tables from six commercial child day-care centers were sampled

during a 5-month period. Since recoveries from fiberglass surfaces

were not reliable in our laboratory trials, we enhanced potential

cyst recoveries by sampling, as previously described, five 50 cm2 areas from each randomly selected fiberglass chair. For consis-tency, table surfaces were similarly sampled. The five samples from each test surface were pooled, sealed with Parafilm#{174}, refrig-erated, processed, and examined within I month.

Giardiasis

Survey

RESULTS

Thirty-five percent of the potential target population (80!231)

of 2- to 3-year-old children and their care givers participated in

TABLE 1. Recovery of G. lamblia Cys ts From Surfaces

Cysts in Surface Surface Recoveries

Inoculum (Number) Area (cm2) Composition . Individual Mean

190 50 Formica#{174} 70, 50, 78 35

50 50 Formica#{174} 21, 38, 31 53

20 50 Formica#{174} 7, 6, 5 30

10 50 Formica#{174} 2, 5, 3 33

20 400 Formica#{174} 3, 6, 13 37

10 400 Formica#{174} 0, 1, 3 13

186 400 Stainless steel 38, 11 13

190 400 Wood 0, 0, 0 0

190 400 Fiberglass 0, 0, 0 0

this study. Sixteen percent of the participants were infected with G. latnblia (I3!80) (Table 2). Only one of the six centers did not have a child with G. lamblia-positive sample (Table 2). Seven of the 13 positive children had diarrhea.

No samples from care givers were positive for

C. lamblia

cysts, although two had children who were positive. Five family mem-bers from four different families were later found to be infected with

G.

lamblia: two fathers, one mother, and two siblings. The siblings attended the same day-care center but were not in the original age-group tested. Six additional children were tested for

G. lamblia

because classmates were infected; all six tests were negative.

For the experienced investigator, light and fluorescent micros-copy were equally effective in detecting C. lamblia. However, detection of G. lamblia by fluorescent microscopy proved to be

more reliable than detection by light microscopy when trained,

but inexperienced, students performed the examinations. Of 85 samples, students detected 15 positive samples using fluorescent microscopy, compared with I I positive samples using light mi-croscopy. Retrospective examination of all individual specimens by light and fluorescent microscopy showed that no false nega-tives resulted from pooling specimens.

Testing of Environmental Surfaces

Laboratory

Development

of Method

Cyst recoveries averaged 30% to 53% of inoculum from 50-cm2 Formica#{174} surfaces, without false negatives (Table 1). Formica#{174} recoveries averaged 13% to 37% of inoculum for standards in the range of 10 to 20 cysts!400 cm2, with 17% false negatives. These results show that Giardia cysts are reliably recovered from For-mica#{174}surfaces, even with low levels of contamination over a large surface area. Average cyst recovery from 400-cm2 stainless steel surfaces was 4% to 13%, with 30% false negatives (Table 1); analyses were not made at 50 cm2. Cysts were not recovered from

400-cm2 wood and rough fiberglass surfaces; analyses were not made at 50 cm2.

Field

Results

Of 53 chairs and tables examined in six commercial child day-care centers, two fiberglass chairs (6%) and one Formica#{174} table surface (2%) were positive for cysts of G. lamblia. Positive results

were from two centers.

DISCUSSION

The 16% infection rate and the percentage of G.

lamblia-positive

children who did not exhibit diarrhea are consistent with earlier reports.”’59 Further, finding only one of the six centers without a

G.

lamblia-positive child also agrees with earlier reports.2 As reported in other studies,5 care givers were not infected.

However, two care givers each had one infected child attending the day-care center in which they worked. This suggests that careful personal hygiene by care givers is not enough to prevent infection in children. As in other studies, some family members

were infected, suggesting possible transmission at home as well as

within child-care centers 3,10

Detection of G. lamblia was slightly more reliable when trained, but inexperienced, students performed the examinations using

fluorescent microscopy instead of light microscopy. Although this difference is not statistically significant (P < .05), missing con-firmed positive specimens has potential clinical significance. As in

the study by Wahlquist and colleagues,” pooling of specimens did

not result in false negatives.

TABLE 2. Child Partici pation and Infec tion Rates by Center

Center Potential Actual Participants With

Participants Participants G. lamblia

# (%)

1 25 8 1(13)

2 59 22 0(0)

3 45 25 8(32)

4 27 6 1(17)

5 50 17 2(12)

6 25 2 1(50)

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1008 SUPPLEMENT

Previous studies of surface sanitation in child day-care centers

have focused on bacterial contamination.67 This is the first

re-ported successful method of examining environmental surfaces for diarrhea-causing protozoan cysts. Discovery of G. lamblia cysts on environmental surfaces shows a potential for transmitting

dis-ease, although we did not determine cyst viability in this study.

Surfactant enhances recovery of Giardia cysts from filters.’2 In our recovery procedure, surfactant also enhanced recovery of cysts from a variety of surfaces. These findings suggest that soap or detergent would help remove potentially infectious cysts as well as dirt from surfaces. Preliminary cleaning with a surfactant (soap or detergent), particularly of heavily soiled surfaces, may reduce the hazard of cyst contamination on surfaces in child-care centers. These observations are consistent with recommendations of the Symposium on Infectious Diseases in Child Day Care.’3

Both the amount and type of solution used to clean surfaces are important. Test results from Formica#{174} surfaces showed greater

recoveries when I mL of PBS-B was used to swab 50 cm2 than

when I mL of PBS-B was used to swab 400 cm2 (Table 1). Although

we did not recover cysts from fiberglass during laboratory trials (I mL of PBS-B/400 2), we did recover cysts from fiberglass chairs

during field trials when we pooled samples from five separate

areas (5 mL of PBS-B/250 cm2). This further suggests that greater

cyst removal can be expected when surfaces are more heavily wetted with surfactant-containing cleaning solutions.

Results of this study suggest that effective cleaning to mechan-ically remove cysts from surfaces followed by sanitizing is an important part of Giardia infection control in child day-care cen-ters. Excluding children from day-care is expensive and does not

eliminate Giardia infection from the centers.4 One reason for the limited effectiveness of exclusion policies may be that infectious cysts remain on surfaces even when infected children are excluded from the day-care environment. Effective personal hygiene has been shown to reduce the incidence of infectious disease in

child-care centers.’ Effective cleaning of environmental surfaces could

be another inexpensive measure to control Giardia infection.

ACKNOWLEDGMENTS

This work was supported by grants from the Georgia Health

Foundation, the Foodservice & Packaging Institute, and the

Geor-gia State University Small Grants Program. Specialty reagents

were donated by Meridian Diagnostics, Inc. KinderCare Learning

Centers served as a major sampling site.

We gratefully acknowledge the assistance of graduate students

Carlton Bessinger, Banks Helfrich, Sara Hinkle, Deborah Jackson,

Page Love, Robin Memman, and Guity Yamin, who collected and analyzed specimens, aided by Steve Martin, laboratory technician.

REFERENCES

1. Black E, Dykes AC, Anderson KE, et aI. Handwashing to prevent diarrhea in day-care centers. Am IEpidemiol. 1981;113:445-451

2. Pickering, LK, Woodward WE, DuPont HL, Sullivan P. Occurrence of Giardia la,nblia in children in day care centers. I Pediatr. 1984;104:522-526 3. Klein JO. Infectious diseases and day care. Ret’ Infect Dis. 1986;8:521-526 4. Bartlett AV, Englender SJ, Jarvis Ba, Ludwig L, Carlson, JF, Topping

JP. Controlled trial of Giardia lamblia: control strategies in day care centers. Am J Public Health. 1991;81:1001-1006

5. Sealy DP, Schuman SH. Endemic giardiasis and day care. Pediatrics.

1983;72:154-158

6. Cody M, O’Leary V. Foodservice Sanitation in Commercial Daycare Centers (abstract). Annual meeting of the Institute of Food Technologists: New Orleans, 1988

7. Holaday B, Pantell R, Louis C, Gilliss CL. Patterns of fecal coliform contamination in day-care centers. Public Health Nursing. 1990;7:224-228 8. Wolfe MS. Symptomatology, diagnosis, and treatment. In: Erlandsen

SL, Meyer EA, eds. Giardia and Giardiasis: Biology, Pathogenesis, and Epidemiology. New York: Plenum Press; 1984

9. Novotny TE, Hopkins RS, Shillam P, Janoff EN. Prevalence of Giardia lamblia and risk factors for infection among children attending day-care facilities in Denver. Public Health Rep. 1990;73:72-75

10. Pickering LK, Woodward WE. Diarrhea in day care centers. Pediatr

Infect Dis. 1982;1 :47-52

11. Wahiquist SP, Williams RM, Bishop H, et al. Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia. IClii, Micro. 1991; 29:1725-1726

12. Payment P, Berube A, Perreault D, Armon R, Trudel M. Clostridium perfringens, human enteric viruses, and coliphages from large volumes of drinking water, using a single filtration. Can IMicrobiol. 1989;35: 932-935

13. Aronson 55, Osterholm MT. Infectious diseases in child day care man-agement and prevention: summary of the symposium and recommen-dations. Rev Infect Dis. 1986;8:672-679

The

Frequency,

Level,

and

Distribution

of

Fecal

Contamination

in

Day-Care

Center

Classrooms

Danielle

J.

Laborde,

MPH,

PhD*;

Kristen

A.

Weigle,

MD,

MPH*;

David

J.

Weber,

MD,

MPH*;

Mark

D. Sobsey,

PhD;

and

Jonathan

B. Kotch,

MD,

MPH

Children in day care are at higher risk of infectious diarrhea than those who remain at home.’ Diarrhea in day care centers (DCCs) is propagated through the fecal-oral route,3’4 a mode of transmission that is enhanced by fecal contamination. Knowledge of the pattern and degree of fecal contamination in DCC class-rooms can identify important sources of enteropathogens to be targeted by hygienic interventions. This study was undertaken to qualitatively and quantitatively describe fecal contamination in DCC classrooms and to determine the relative stability of patterns of contamination across time.

Fecal coliforms occur in large numbers in human feces and can

be presumptively identified on differential culture media. Each discrete colony represents a bacterial cell originally present on the surface that was sampled. In principle, the number of fecal colif-orms rather than the frequency of positive samples reported in

From the Departments of *Epidemiology, Environmental Science and Engineering, and §Maternal and Child Health, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC.

The use of trade names is for identification only and does not constitute endorsement by the Public Health Service, the Centers for disease Control and Prevention, or any of the other co-sponsors of this conference.

previous DCC studies5’6 should more accurately reflect entero-pathogen characteristics of infectious dose and survival in the environment. Therefore, fecal contamination was measured by the number of viable fecal coliforms recovered from environmental surfaces and the hands of children and DCC staff members. The distribution of fecal contamination was described in terms of coliform presence and coliform density (counts) for individual classroom sites.

METHODS

Fecal contamination was measured during two prospective studies in North Carolina. In study I, 13 Cumberland County DCCs served as controls during a hygienic intervention trial and were sampled from April 1989 to March 1990; in study 2, 25 Research Triangle Area DCCs were sampled between January and August 1991. Cumberland County includes three large com-munities: Fayetteville, Fort Bragg, and Pope Air Force base. The Research Triangle Area includes Durham, Orange, and Wake counties.

We monitored fecal contamination in classrooms for infants (those providing care for children under 6 months of age) and in classrooms for toddlers (those that did not provide care for

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1994;94;1006

Pediatrics

Mildred M. Cody, Henry M. Sottnek and Virginia S. O'Leary

Cysts From Chairs and Tables in Child Day-Care Centers

Giardia lamblia

Recovery of

Services

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1994;94;1006

Pediatrics

Mildred M. Cody, Henry M. Sottnek and Virginia S. O'Leary

Cysts From Chairs and Tables in Child Day-Care Centers

Giardia lamblia

Recovery of

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