Animal and human dose-response models as well as in vitro cell culture have been widely used to evaluate
Numerous cross-transmission experiments involving the infection with isolates of
Hence the main objective of the present study was to develop recommendations for the standardization of animal dose-response experiments by conducting a meta-analysis of individual subjects and exploring study design characteristics that cause heterogeneity between included studies. The hypothesis tested in this meta-analysis was that selected experimental factors of interest are associated with increased
The search of published animal and/or human dose-response studies in all languages was performed using the electronic databases PubMed and Web of Science from March to December, 2010. The process is described in Figure
Flow of information through the different phases of a systematic literature review for the
The articles identified by means of these search criteria were subject to a further selection process consisting of the removal of the complete study or individuals within the articles that met any of the exclusion criteria (Table
Exclusion criteria for studies and number of articles excluded from the
Exclusion criteria | Articles excluded |
---|---|
(1) |
19 |
(2) Infection was assessed only by histology on tissue sections | 6 |
(3) Number of animals with diarrhoea or shedding was not reported | 4 |
(4) Animal study groups were subject to treatments other than the single inoculation/single outcome design used in this meta-analysis | 3 |
(5) Infection was assessed by cyst shedding and histology together and it was not possible to distinguish how many animals were shedding | 4 |
(6) Experimental dose response was measured in humans only | 2 |
(7) Study was not an experimental dose response experiment | 1 |
(8) Cyst dose administered to the experimental host was not provided or a range was provided | 1 |
(9) Number of animals used was not provided | 1 |
Data regarding the following variables for each individual animal were extracted and recorded from each article: (i)
The unit of analysis was the individual animal; thus, the value for each variable was collected for each animal and included in the analysis. If a variable was not reported at the animal level, the variable was left blank. Table
Studies included for
Reference | Year | Outcome | Sample size (number of animals) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
IS1 | P2 | ST3 | ES4 | A5 | IS6 | GD7 | AR8 | DM9 | |||
[ |
1978 | S10 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
[ |
1979 | S10 | 47 | 47 | NR11 | 47 | 47 | 47 | 47 | 47 | NR11 |
[ |
1982 | S10 | 11 | 11 | 11 | 11 | 11 | 11 | 11 | 11 | NR11 |
[ |
1982 | S10 | 150 | 150 | 150 | 150 | 150 | 150 | 150 | 150 | 150 |
[ |
1984 | S10 | 49 | 49 | NR11 | 49 | 49 | 49 | 49 | 49 | NR11 |
[ |
1985 | S10 | 25 | 25 | 14 | 25 | NR11 | 25 | 25 | 25 | 25 |
[ |
1986 | S10 | 60 | 60 | NR11 | 60 | 49 | 60 | 60 | 60 | 60 |
[ |
1988 | S10 | 204 | 204 | 107 | 204 | 204 | 204 | 204 | 204 | 204 |
[ |
1989 | S10 | 10 | 10 | NR11 | 10 | NR11 | 10 | 10 | 10 | 10 |
[ |
1990 | S10 | 75 | 75 | NR11 | 75 | 75 | 75 | 75 | 75 | 75 |
[ |
1991 | S10 | NR11 | NR11 | NR11 | 10 | 10 | 10 | 10 | 10 | 10 |
[ |
1991 | S10 | 109 | 109 | 109 | 109 | 109 | 109 | 109 | 109 | 109 |
[ |
1992 | S10 | NR11 | 14 | NR11 | 14 | 14 | 14 | 14 | 14 | 14 |
[ |
1993 | S10 | 62 | 62 | NR11 | 62 | 62 | 62 | 62 | 62 | 62 |
[ |
1994 | S10 | 4 | 16 | NR11 | 16 | 16 | 16 | 16 | NR11 | 16 |
[ |
1996 | S10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
[ |
1997 | S10 | NR11 | 10 | NR11 | 10 | 10 | 10 | 10 | 10 | 10 |
[ |
2002 | S10 | NR11 | 94 | NR11 | 94 | 94 | 94 | 94 | 94 | 94 |
[ |
2005 | S10 | 56 | 56 | NR11 | 56 | 56 | 56 | 56 | 56 | 56 |
[ |
2006 | S10 | NR11 | 6 | NR11 | 6 | 6 | 6 | 6 | 6 | 6 |
[ |
2007 | S10 | 97 | 97 | 97 | 97 | 97 | 97 | 97 | 97 | 97 |
[ |
2007 | S10 | 6 | 6 | NR11 | 6 | 6 | 6 | 6 | 6 | 6 |
[ |
2008 | S10 | NR11 | 12 | NR11 | 12 | 12 | 12 | 12 | 12 | 12 |
[ |
2008 | S10 | NR11 | 60 | NR11 | 60 | 60 | 60 | 60 | 60 | 60 |
[ |
2010 | S10 | 40 | 40 | NR11 | 40 | 40 | 40 | 40 | 40 | 40 |
[ |
2010 | S10 | 33 | 33 | 33 | 33 | 33 | 33 | 33 | 33 | 33 |
[ |
1995 | S and D12 | 7013, 2814 | 7013, 2814 | 3013, 814 | 7013, 2814 | 7013, 2814 | 7013, 2814 | 7013, 2814 | 4013, 2014 | 7013, NA15 |
[ |
1995 | S and D12 | 5213, 3014 | 5213, 3014 | 2213, NR11 | 5213, 3014 | 5213, 3014 | 5213, 3014 | 5213, 3014 | 5213, 3014 | 5213, NA15 |
[ |
1997 | S and D12 | 1016 | 1016 | NR11 | 1016 | 1016 | 1016 | 1016 | 1016 | NA15 |
[ |
2010 | S and D12 | 1416 | 1416 | NR11 | 1416 | 1416 | 1416 | 1416 | 1416 | NA15 |
The extracted variables were classified according to the information provided by the selected studies for each outcome (Table
Classification of categorical variables for both
Variable | Shedding outcome | Diarrhoea outcome | ||||
---|---|---|---|---|---|---|
Classification of variables1 | Number of studies | Number of individuals | Classification of variables1 | Number of Studies | Number of individuals | |
Assemblage | A | 3 | 148 | A and E | 1 | 14 |
B | 1 | 49 | ||||
E | 1 | 6 | ||||
A and E | 1 | 14 | ||||
|
||||||
Isolate Source | Humans |
19 |
1128 |
Humans |
1 |
28 |
|
||||||
Passage | No |
17 |
755 |
No |
2 |
24 |
|
||||||
Storage Time | <1 week |
10 |
420 |
<1 week | 1 | 8 |
|
||||||
Experimental Species | Mice |
7 |
180 |
Other Animals |
2 |
24 |
|
||||||
Age | Adult |
12 |
458 |
Newborn |
2 |
24 |
|
||||||
Infective Stage | Cysts |
20 |
1021 |
Cysts |
3 |
64 |
|
||||||
Administration Route | Gastric Intubation |
6 |
233 |
Gastric Intubation |
2 |
50 |
|
||||||
Detection Method | Hemocytometer |
15 |
749 |
Not analyzed |
Criteria used to determine age categories for experimental species (alphabetical order).
Animal species | Criteria used | ||
---|---|---|---|
Newborn | Weanling1 | Adult | |
Cattle | <21 weeks | 21–64 weeks [ |
≥65 weeks [ |
Cats | <8 weeks | 8–25 weeks [ |
≥26 weeks [ |
Dogs | <8 weeks | 8–29 weeks [ |
≥30 weeks [ |
Gerbils | <4 weeks | 4–7 weeks [ |
≥8 weeks [ |
Hamsters | <3 weeks | 3 weeks [ |
>3 weeks |
Mice | <3 weeks | 3 weeks [ |
>3 weeks [ |
Rabbits | <4 weeks | 4 weeks–17 weeks [ |
≥18 weeks [ |
Rat | 3 weeks | 3–7 weeks [ |
≥8 weeks[ |
In this meta-analysis, the statistical analysis and publication bias assessment were done as described in Adell et al. [
As shown in Figure
The bivariate analysis showed that the covariates “Passage,” “Experimental Species,” “Age,” “Infective Stage,” and “
Bivariate analysis results for risk factors associated with
Variable1 |
|
Statistical association between variable and cyst shedding (Yes/No)2 |
---|---|---|
Passage | <0.00013 | Yes |
Storage Time | 0.92693 | No |
Experimental Species | <0.00013 | Yes |
Age | 0.00233 | Yes |
Infective Stage | <0.00013 | Yes |
|
0.00614 | Yes |
2Significant level ≤0.2.
3Mantel-Haenzel bivariate analyses analysis taking into account the correlation between studies.
4GLIMMIX bivariate analyses.
Multivariable generalized linear mixed model showing risk factor associations for
Variable | Categories | Odds ratio estimate | 95% Wald confidence limits |
|
---|---|---|---|---|
Experimental Species | Gerbils2 | |||
Mice | 0.163 | (0.003, 8.87) | 0.37 | |
Other Animals | 0.034 | (<0.001, 5.05) | 0.18 | |
Other Rodents | 4.305 | (0.10, 183.05) | 0.45 | |
|
||||
Age | Young2 | |||
Adult | 0.36 | (0.05, 2.43) | 0.30 | |
|
||||
Infective Stage | Trophozoites2 | |||
Cysts | 5.026 | (2.63, 9.56) | <0.00112 | |
|
||||
|
Not applied (continuous variable) | 2.677 | (1.81, 3.94) | <0.00112 |
|
||||
Administration Route | Gastric Intubation2 | |||
Other Than Gastric Intubation | 10.39 | (0.19, 573.61) | 0.25 | |
|
||||
|
Mice | 4.368 | (1.59, 11.93) | 0.00412 |
Other Animals | 2.129 | (0.12, 37.45) | 0.61 | |
Other Rodents | 0.0910 | (0.01, 0.72) | 0.0212 | |
|
||||
|
Cysts | 0.5711 | (0.41, 0.81) | 0.00212 |
1Generalized chi-square statistic and its degrees of freedom (gener. Chi-sq/df) = 0.66.
2Corresponds to the reference category.
3The odds ratio estimate corresponds to Mice versus Gerbils at the mean log dose.
4The odds ratio estimate corresponds to Other Animals versus Gerbils at the mean log dose.
5The odds ratio estimate corresponds to Other Rodents versus Gerbils at the mean log dose.
6The odds ratio estimate corresponds to Cysts versus Trophozoites at the mean log dose.
7The odds ratio estimate corresponds to an increase of one in log dose in Gerbils with Trophozoites.
8The odds ratio corresponding to an increase of one in log dose in Mice is 4.36 times that in Gerbils.
9The odds ratio corresponding to an increase of one in log dose in Other Animals is 2.12 times that in Gerbils.
10The odds ratio corresponding to an increase of one in log dose in Other Rodents is 0.09 times that in Gerbils.
11The odds ratio corresponding to an increase of one in log dose in Cysts is 0.57 times that in Trophozoites.
12Risk factors with statistically significant results.
The multivariable model indicated that inoculating cysts into the experimental host had 5.02 times higher odds of cyst shedding than inoculating trophozoites at the mean log dose of 0.0538 (
The model also indicated that, for each unit of increase in the log dose administered to “Mice,” the odds of cyst shedding increased 4.36 times compared to “Gerbils” (
Range of
The fit of the model was assessed by the ratio value for generalized chi-sq/df value and residual plots. The generalized chi-sq/df for the multivariable model was 0.66, indicating that the model had good fit. The residual plots (not shown), in which the residual values were plotted against the linear predictor, showed that two observations in each model had large residuals (≥15) and were possible outliers from the cluster of observations. Removing the individuals that had large residuals did not change the significance of the other variables of the model; thus, they were kept in the model.
In this meta-analysis, we found evidence of possible publication bias for the cyst shedding outcome. The funnel plot showed a higher number of studies over the mean value (Figure
Funnel plot of standard error by logit event rate for cyst shedding outcome after
As the data for the diarrhoea outcome were sparse, the analysis done for the shedding outcome could not be performed for the diarrhoea outcome. Therefore, the bivariate analysis was done considering the data as a pool across studies to estimate associations rather than accounting for the correlation between studies as done for the shedding outcome. The bivariate analysis showed that, for the outcome of diarrhoea in exposed animals, the covariates “Isolate Source” and “Experimental Species” were significant (
Bivariate analysis results for risk factors associated with diarrhoea outcome after
Variable1 |
|
Statistical association between variable and presence of diarrhoea (Yes/No)2 |
---|---|---|
Isolate Source | <0.00013 | Yes |
Passage | 0.353 | No |
Experimental Species | 0.073 | Yes |
Age | 0.353 | No |
Infective Stage | 0.253 | No |
|
0.254 | No |
Administration Route | 0.323 | No |
2Significant level ≤0.2.
3Mantel-Haenzel bivariate analyses not taking into account the correlation between studies.
4GLIMMIX bivariate analyses.
This meta-analysis evaluated the effects of multiple experimental covariates on cyst shedding or diarrhoea as indicators of
Based on the results of this meta-analysis, it would be appropriate to make the following recommendations for future dose-response experiments on
In the case of assessing infection by means of the presence of diarrhoea in experimentally infected animals, more experimental studies in animal models should be conducted, as not enough studies have been reported to obtain estimates of the effect of different experimental parameters on diarrhoea in individual animals. It should be noted that while the diarrhoea outcome is of clinical relevance, the presence of asymptomatic infected individuals is a limitation of using diarrhoea to represent infectious status. Based on the bivariate analyses, it would be appropriate to consider and report the following in future dose-response experiments: (i) the assemblage being inoculated into the experimental host, (ii) original source of the cysts being inoculated, (iii) whether cysts or trophozoites were subject to any passage before inoculation into the experimental host or not, (iv) animal species used as experimental hosts, (v) age of the experimental host, and (vi) the administration route used to inoculate the cysts or trophozoites into the experimental host. These studies will provide better information and more comparable results for risk assessments that consider illness as an outcome.
Interestingly, the significant “
Pathogen shedding patterns for newborns, weanlings, and adults can be quite different across host species. For instance, it has been reported that weanling calves generally lack a strong specific humoral immune response to
The multivariable model suggested that inoculating the experimental host with cysts would increase the likelihood of cyst shedding compared to inoculation with trophozoites. However, the model also indicated that an increase of one log in dose had less of an impact on cysts than it did on trophozoites. Therefore, differences by using cysts versus trophozoites for host infection depend on the Giardia dose used, and it is advisable to consider these two variables together when designing experimental studies. Results of the multivariable model also suggest that when a higher dose of trophozoites is administered to gerbils, the odds of cyst shedding increase. Numerous studies in which different doses of Giardia cysts or trophozoites have been administered to animal models have been published. However, whether the infectious dose may contribute to symptom variability is still unclear [
Information for the variables “Assemblage,” “Isolate Source,” and “Storage Time” was scarce and thus the association between these variables and the shedding outcome could not be assessed. Passage of cysts or trophozoites used to infect experimental hosts was identified as a risk factor for cyst shedding in the bivariate analysis. However, information on this variable was not reported for all studies, which made it challenging to consider it jointly in a multivariable model. Once more experimental studies providing information regarding these variables are published, it would be beneficial to evaluate the association with the shedding outcome and incorporate them in the multivariable model “Experimental Species,” “Age,” “Infective Stage,” and “
This meta-analysis showed evidence of possible publication bias for the cyst shedding outcome. This finding can be explained by the fact that smaller studies are more likely to be published if they have larger than average effects, which makes them more likely to meet the criterion for statistical significance [
Based on the results of this meta-analysis, it is crucial that more experimental studies in animal models are conducted to assess infectivity of
When assessing
Young and adult animals were similarly likely to shed cysts. Nevertheless, additional studies are needed for increased statistical power to ascertain effects of the log dose increment for different age groups on the presence of cyst shedding.
For considering whether cysts or trophozoites are used to challenge the experimental hosts to assess infection by means of cyst shedding, the multivariable analysis results suggest that it would be more appropriate to use cysts. However, it also indicated that an increase of one log dose has less of an impact for cysts than for trophozoites; thus, differences between inoculating cysts or trophozoites in the cyst shedding depend on the dose administrated. As expected, administering higher doses of cysts or trophozoites increases the odds of cyst shedding.
When using a diarrhoea outcome measure in experimental studies, the source of the isolate and species of experimental animal host should be considered when designing experimental studies. As additional studies are published, greater power will be possible to distinguish individual and joint effects of the identified covariates.
The authors declare that there is no conflict of interests regarding the publication of this paper.
We would like to acknowledge the financial support received from the “Presidente de la República” Chilean scholarship, the Central Coast Long-term Environmental Assessment Network (CCLEAN) (Grant no. 06-076-553) from the California State Water Board to the City of Watsonville, and the National Science Foundation (NSF) Ecology of Infectious Disease Grant Program (Grant no. OCE-1065990). Thanks are due to Fernando O. Mardones and Francisco Puentes for comments and suggestions.