## General overview

*Acanthamoeba* spp. are free-living amoeba (FLA) that have been commonly found in freshwater, tap water, and recreational water. *Acanthamoeba* spp. are capable of causing a variety of infections, including *Acanthamoeba* keratitis, an eye infection that has been associated with contect lens usage and cornea damage, and Granulomatous Amebic Encephalitis (GAE), a serious central nervous system infection that primarily affects immunocompromised individuals (Marciano-Cabral & Cabral, 2003; Visvesvara, Moura, & Schuster, 2007). Considering these infections, some exposure routes of concern for *Acanthamoeba* spp. are the corneal and intranasal exposure routes.

Dean et al. (2020) fit dose response models to data from previously conducted animal studies for *Acanthamoeba* spp. and the corneal and instranasal exposure routes. These models are an important step towards characterizing the risk associated with FLA like *Acanthamoeba* for drinking water-relevant exposure scenarios. More detailed descriptions of the datasets, fitting methods, model evaluation, and results can be found in the published article: http://dx.doi.org/10.1111/risa.13603

## Recommended Model

For the intranasal exposure route, the exact beta-Poisson model fit to the pooled data from Experiments 3 and 4 is the recommended model. The successful pooling of the model gives additional confidence for applying the dose response relationship to additional *Acanthamoeba* strains and species.

Dean, K., Tamrakar, S., Huang, Y., Rose, J.B., & Mitchell, J. (2020). Modeling the dose repsonse relationship of waterborne Acanthamoeba. *Risk Analysis. *http://dx.doi.org/10.1111/risa.13603

Marciano-Cabral, F., & Cabral, G. (2003). *Acanthamoeba* spp. as agents of disease in humans. *Clinical Microbiology Reviews*. https://doi.org/10.1128/CMR.16.2.273-307.2003

Visvesvara, G.S., Moura, H., & Schuster, F.L. (2007). Pathogenic and opportunistic free-living amoebae: *Acanthamoeba* spp., *Balamuthia mandrillaris*, *Naegleria fowleri*, and *Sappinia diploidea*.* FEMS Immunology & Medical Microbiology, 50*: 1-26.

ID | # of Doses | Agent Strain | Dose Units | Host type | Μodel | Optimized parameters | Response type | Reference |
---|---|---|---|---|---|---|---|---|

Acanth_Intranasal2 | 3 | A. castellanii HN-3 | no of trophozoites | mice | exponential |
k = 1.31E-04 |
death | |

Acanth_Intranasal3 | 3 | A. castellanii HN-3 | no of trophozoites | mice | exponential |
k = 8.54E-04 |
brain invasion | |

Acanth_Intranasal4 | 3 | A. castellanii HN-3 | no of trophozoites | mice | exponential |
k = 2.79E-04 |
acute meningoencephalitis | |

Acanth_Intranasal1 | 6 | A. culbertsoni (A1) | no of trophozoites | mice | beta-Poisson |
a = 0.161 N _{50} = 14,690 or 14,538 |
death | |

Acanth_Intranasal_Pooled | 9 | A. castellanii HN-3 and A culbertsoni A1 | no of trophozoites | mice | beta-Poisson |
a = 0.245 N _{50} = 19357 (beta=1215) |
death |

k = 1.31E-04

Culbertson et al. (1966) studied the pathogenicity of the HN-3 strain of *A. castellanii *(Culbertson et al., 1966; Marciano-Cabral & Cabral, 2003) on ether-anesthetized-specific-pathogen-free (SPF) mice. Cultures of amebae were grown in trypticase soy broth and diluted so that 0.03 mL of a concentrated suspension could be instilled intranasally into the mice by placing fluid over the nares (Culbertson et al., 1966; Culbertson, Ensminger, & Overton, 1965a; Culbertson, Ensminger, & Overton, 1965b).

The exponential model provided the best fit to the data.

Culbertson, C. G., Holmes, D. H., & Overton, W. M. (1965b). Hartmanella castellani (Acanthamoeba sp.). The American Journal of Clinical Pathology, 43(4), 361–364.

Culbertson, C. G., Ensminger, P. W., & Overton, W. M. (1966). Hartmannella (Acanthamoeba): Experimental Chronic, Granulomatous Brain Infections Produced by New Isolates of Low Virulence. The American Journal of Clinical Pathology, 46(3), 305–314.

k = 8.54E-04

Culbertson et al. (1966) studied the pathogenicity of the HN-3 strain of *A. castellanii* (Culbertson et al., 1966; Marciano-Cabral & Cabral, 2003) on ether-anesthetized-specific-pathogen-free (SPF) mice. Cultures of amebae were grown in trypticase soy broth and diluted so that 0.03 mL of a concentrated suspension could be instilled intranasally into the mice by placing fluid over the nares (Culbertson et al., 1966; Culbertson, Ensminger, & Overton, 1965a; Culbertson, Ensminger, & Overton, 1965b).

The exponential model provided the best fit to the data.

**Figure 1: Plot of the exponential model fit to Experiment 5 with upper and lower 95% and 99% confidence**

**Figure 2: Histogram of the 10,000 bootstrap replicates of k for the best fitting exponential model fit to Experiment 5**

Culbertson, C. G., Holmes, D. H., & Overton, W. M. (1965b). Hartmanella castellani (Acanthamoeba sp.). The American Journal of Clinical Pathology, 43(4), 361–364.

Culbertson, C. G., Ensminger, P. W., & Overton, W. M. (1966). Hartmannella (Acanthamoeba): Experimental Chronic, Granulomatous Brain Infections Produced by New Isolates of Low Virulence. The American Journal of Clinical Pathology, 46(3), 305–314.

k = 2.79E-04

Culbertson et al. (1966) studied the pathogenicity of the HN-3 strain of *A. castellanii *(Culbertson et al., 1966; Marciano-Cabral & Cabral, 2003) on ether-anesthetized-specific-pathogen-free (SPF) mice. Cultures of amebae were grown in trypticase soy broth and diluted so that 0.03 mL of a concentrated suspension could be instilled intranasally into the mice by placing fluid over the nares (Culbertson et al., 1966; Culbertson, Ensminger, & Overton, 1965a; Culbertson, Ensminger, & Overton, 1965b).

The exponential model provided the best fit to the data.

**Figure 1: Plot of the exponential model fit to Experiment 6 with upper and lower 95% and 99% confidence**

**Figure 2: Histogram of the 10,000 bootstrap replicates of k for the best fitting exponential model fit to Experiment 6**

Culbertson, C. G., Ensminger, P. W., & Overton, W. M. (1966). Hartmannella (Acanthamoeba): Experimental Chronic, Granulomatous Brain Infections Produced by New Isolates of Low Virulence. The American Journal of Clinical Pathology, 46(3), 305–314.

N

_{50}= 14,690 or 14,538

Červa (1967a,b) studied white mice of the Czechoslovak H-strain weighing 13-15 grams inoculated intranasally by placing 0.02 mL of the A1 strain of *Acanthamoeba* over the nares of the ethyl-ether anesthetized mice (Cerva, 1967b).

The beta-Poisson model provided the best fit to the data.

Cerva, L. (1967b). Intranasal, Intrapulmonary, and Intracardial Inoculation of Experimental Animals with Hartmanella castellanii. Folia Parasitologica (Praha), 14, 207–215.

N

_{50}= 19357 (beta=1215)

The same exposure route and endpoint was evaluated for Experiments 3 and 4 (Cerva, 1967b; Culbertson et al. 1966). A pooling analysis was attempted and successful. The beta-Poisson model provided a good fit to the pooled data and is shown below in Figure 1. Note: both the exact and approximate beta-Poisson models were fit to the data. The figures shown below and the csv file of bootstrapped parameter replicates are for the best fitting parameters of the exact beta-Poisson model. The successful pooling of multiple datasets generally increases the confidence in the estimated model parameters.

**Figure 1: Plot of the beta-Poisson model fit to the pooled Experiments 3 and 4 with upper and lower 95% and 99% confidence**

**Figure 2: Uncertainty plot of the 10,000 paired bootstrap replicates of alpha and beta for the pooled beta-Poisson model. **

Cerva, L. (1967b). Intranasal, Intrapulmonary, and Intracardial Inoculation of Experimental Animals with *Hartmanella castellanii*. *Folia Parasitologica (Praha)*, *14*, 207–215.

Culbertson, C. G., Ensminger, P. W., & Overton, W. M. (1966). *Hartmannella* (*Acanthamoeba*): Experimental Chronic, Granulomatous Brain Infections Produced by New Isolates of Low Virulence. *The American Journal of Clinical Pathology*, *46*(3), 305–314.