ASYMPTOMATIC CARRIAGE OF PROTOZOAN PARASITES IN CHILDREN IN ... : The Pediatric Infectious Disease Journal (original) (raw)
_Cryptosporidium_and Giardia are the most important causes of protozoal gastroenteritis in the United Kingdom. Most of the human Cryptosporidium infections in England and Wales are caused by 2 species, Cryptosporidium hominis and Cryptosporidium parvum, and other species/genotypes are found much less commonly.1
A large-scale case-control study of sporadic cases in the United Kingdom found that changing diapers was an independent risk factor for infection with C. hominis (ORadj 4.0, P < 0.001), even if the child did not have diarrhea.2 The authors speculated that asymptomatic carriage in young children might be common and an important reservoir of infection. However, few studies of asymptomatic carriage in children have been carried out in the developed world. Carriage rates of 1.8% to 3.8% have been found in cross-sectional studies of immunocompetent children in child-care settings in the United States, France, and Spain.3 One limitation of these studies was the use of staining and microscopy, with or without concentration, to detect Cryptosporidium oocysts, a relatively insensitive method. Greatly improved sensitivity has been demonstrated by immunomagnetic separation (IMS) modified for human stool samples,4 which can detect as few as 2 oocysts per gram (opg) of feces. Low numbers of organisms are clinically as well as epidemiologically important, since the infectious dose of C. hominis can be as low as single figures.5
Asymptomatic carriage of Giardia is considered to be quite common, even in industrialized countries such as the United States where rates of up to 26% have been found in asymptomatic children in day care facilities.6 In New Zealand, clinical cases of giardiasis were more common in patients who had changed children's diapers (ORadj, 3.8, P < 0.0001),7 very similar to the findings in the United Kingdom for Cryptosporidium.2 It is not known whether this is an important reservoir for the organism in the United Kingdom.
To more accurately determine the carriage rate of Cryptosporidium using IMS and to define the species/genotypes present, we sampled asymptomatic children in preschool day care facilities in the United Kingdom. Samples were also tested for the presence of Giardia sp.
MATERIALS AND METHODS
Study Design, Recruitment, and Sample Collection
This was a community study of infants and preschool children using diapers, recruited from preschool day care facilities in the City of Swansea (population 250,000; location south-west Wales) and the London Borough of Wandsworth (population 280,000; location inner London, with wide ethnic diversity).
The main study was carried out between October and December, the peak season for both C. hominis and Giardia infection. ‘Parent packs' including a self-completion questionnaire, a stool sample pot, and a Royal Mail Safepack were distributed to each participating facility. Only 1 sample was collected per child.
Samples and completed questionnaires were returned unfixed by first-class post to the United Kingdom Cryptosporidium Reference Unit in Swansea. Samples were then stored at 2° to 8°C until processing. Data were analyzed using EpiInfo (Version 6, Centers for Disease Control and Prevention, Atlanta, GA). Differences between the characteristics of the respondents were compared using the χ2 or Fisher exact test. For prevalence estimates, 95% confidence intervals were calculated using the binomial distribution. The same study methodology was used at the 2 centers and the 95% CI for the prevalence estimates overlap considerably, and so data for the 2 centers are combined.
The study was approved by the Dyfed-Powys NHS Research Ethics Committee
Laboratory Methods
Cryptosporidium Detection.
For each sample, 1.5 to 2 g was processed by IMS as described previously.4 Concentrates (110 μL) were split 50:50, for immediate examination by immunofluorescence microscopy (Crypto-Cel, TCS Bioscience Ltd, UK) and for storage at −20°C until molecular analysis. Oocyst concentrations per gram (opg) were calculated. Each sample was tested once, with internal process controls and negative controls included in each run.
Cryptosporidium Species/Genotype Identification.
To identify Cryptosporidium species/genotypes in microscopy positive samples, the remaining IMS concentrate was fractured using 3 cardice freeze/thaw (−80°C/100°C, respectively) cycles prior to purification using a QIAamp DNA extraction kit (Qiagen, Crawley, UK). A nested polymerase chain reaction assay using primers specific for the Cryptosporidium small subunit rRNA (ssu rRNA) gene was used.8 Amplicons were visualized following 2% (wt/vol) agarose gel electrophoresis and Sybr green staining and purified using a QIAquick polymerase chain reaction purification kit (Qiagen, Crawley, UK) prior to sequencing by bidirectional sequence analysis of approximately 830 bp ssu rRNA (GeneService, Essex, UK). Consensus sequences were produced using Chromas Pro (Technylisium, Australia) and compared with GenBank submissions using the Basic Local Alignment Search Tool algorithm (available at: http://www.ncbi.nlm.nih.gov/blast). All sequences generated in this study have been submitted to GenBank under accession numbers FJ031236 to FJ031239.
Giardia Enzyme-Linked Immunosorbant Assay (ELISA).
The Giardia ELISA (Giardia II, Techlab®) was conducted as per the manufacturer's instructions. The test was repeated on all positive samples, for confirmation.
RESULTS
A total of 67 facilities, 31 of 37 (83.8%) in Swansea and 36 of 46 (78.3%) in Wandsworth, agreed to participate and 230 eligible samples were returned for analysis, a return rate from parents of 28%.
The characteristics of children attending the facilities in Swansea and London were broadly similar. The median age of children in the study was 17 months (range: 1–40 months) and 52% had another child living at home. The majority of children had been at nursery for several months and attended a median of 24 hours per week. Three quarters of children were reported as having had a bout of diarrhea at some time in their lives. None had diarrhea at the time of sampling. Two-thirds had been abroad or had visited a farm.
Of the 230 samples returned, 3 (1.3%; 95% CI: 0.3%–3.8%) were positive for Cryptosporidium oocysts by IMS. The isolates were identified as one C. hominis (GP60 subtype IbA10G2, the most prevalent UK subtype9), and 1 each of the Cryptosporidium cervine and skunk genotypes. Oocyst concentrations were 13367 opg (C. hominis), 3471 opg (cervine genotype), and 56 opg (skunk genotype). Similarly, 3 of the 230 samples (1.3%; 95% CI: 0.3%–3.8%) were positive for Giardia by ELISA. There were no dual infections. The characteristics of children from whom Cryptosporidium and Giardia were isolated are shown in Table 1. There were no significant differences between the characteristics of children carrying the parasites and of other children in the study.
Characteristics of Children With Cryptosporidium or Giardia
DISCUSSION
The overall point prevalence of both Cryptosporidium and Giardia carriage in the study was 1.3% (3/230) (95% CI: 0.3%–3.8%). The low response rate could bias the results, if respondents differ systematically from nonrespondents. However, the family composition and characteristics of carriers appear to be typical of study respondents as a whole, and we therefore consider our prevalence estimates to be reasonably robust for this section of the study population.
Despite the use of the more highly sensitive IMS, the prevalence of 1.3% for asymptomatic carriage of Cryptosporidium is similar to or lower than that described in other cross-sectional studies of children in day care.3 Of the 3 positives identified, only 1 contained enough oocysts to make it likely to have been detected by conventional methods.4 Missing the other 2 positives would have given an apparent prevalence of 0.4%.
In our study the prevalence of Giardia was similarly low. The advice of The American Academy of Pediatrics and Centers for Disease Control and Prevention is that since asymptomatic carriage of Giardia is not harmful, and since recolonization is likely after treatment, treatment of asymptomatic cases is generally not indicated.
Of the 3 Cryptosporidium isolates, only 1 was identified as C. hominis, the species responsible for most human-to-human transmission and previously found to be associated with changing diapers.2 The other 2 isolates were the Cryptosporidium cervine and skunk genotypes, which are rarely found in clinical samples.1,10 Therefore, the finding of these genotypes in children in this study was very unexpected and raises the intriguing possibility that such genotypes are circulating more commonly than previously believed, and that asymptomatic carriage of ‘rare' genotypes may be more common than clinical disease caused by them, perhaps reflecting lower virulence in humans, compared with C. hominis and C. parvum. This may be consistent with the lower parasite load in our study for the skunk and cervine genotypes.
In summary, our study does not provide convincing evidence in support of the hypothesis that young children in diapers are a significant source of C. hominis infection in the community.2 The role of asymptomatic carriage in the spread of these infections either within the household or day-care setting remains unclear.
ACKNOWLEDGMENTS
The authors thank Guy Robinson for undertaking molecular analyses and Kristin Elwin, Nigel Crouch, and Stephen Hadfield for liaising with nurseries. The authors also thank the staff of the South West London Health Protection Unit, Sylvia Arrowsmith for assistance with translation, and all the child-care facilities and parents who assisted us in obtaining samples.
REFERENCES
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9. Chalmers RM, Hadfield SJ, Jackson CJ, et al. Geographic linkage and variation in Cryptosporidium hominis. Emerg Infect Dis. 2008;14:496–498.
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Keywords:
Cryptosporidium; Giardia; carriage; day-care facilities; immunomagnetic separation
© 2009 Lippincott Williams & Wilkins, Inc.