A highly sensitive method for detecting Cryptosporidium parvum oocysts recovered from source and finish water using RT-PCR directed to Cryspovirus RNA

Authors: MILENA, SATO DE SOUZA - Sao Paulo State University (UNESP); Obrien, Celia; Santin-Duran, Monica; Jenkins, Mark

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2018
Publication Date: 11/30/2018
Citation: Sato de Souza, M., O'Brien, C., Santin, M., Jenkins, M.C. 2018. A highly sensitive method for detecting Cryptosporidium parvum oocysts recovered from source and finish water using RT-PCR directed to Cryspovirus RNA. Journal of Microbiological Methods. 156:77-80. https://doi.org/10.1016/j.mimet.2018.11.022.
DOI: https://doi.org/10.1016/j.mimet.2018.11.022

Interpretive Summary: Cryptosporidiosis is a diarrheal disease of humans and animals caused by protozoa in the genus Cryptosporidium. The disease is most prominent in young children or immune-compromised individuals, such as those infected with the AIDS virus. Outbreaks of cryptosporidiosis usually stem from ingestion of water contaminated with the highly resistant oocyst stage of the parasite. The parasite is resistant to most standard water disinfectants, can cause clinical infection at very low numbers (~ 130 oocysts in humans), and no effective prophylactic or therapeutic regimen exists that can prevent or ameliorate disease. For this reason, detecting Cryptosporidium oocysts in water is one of the main ways of preventing outbreaks because actions, such as boiled water alerts, can be implemented when oocysts are found. Methods exist for concentrating and examining by microscopy water samples for the presence of Cryptosporidium oocysts, but these techniques suffer from poor sensitivity. In the present study, a molecular biology assay of genetic material (RT-PCR) was developed that targets the internal virus, namely Cryspovirus, of all species of Cryptosporidium, and was used to detect the parasite in water concentrates that had been inoculated with various numbers of C. parvum oocysts. This study found that while protein-based immunofluorscence assays failed to detect low doses of C. parvum in source water, the RT-PCR assay was more sensitive and capable of detecting the parasite. These results suggest that sensitive detection of Cryptosporidium oocysts may require molecular assays, such as the Cryspovirus RT-PCR, in order to prevent outbreaks caused by ingestion of water containing low numbers of oocysts.

Technical Abstract: Sensitive detection of Cryptosporidium oocysts is important because the parasite can cause clinical infection in humans at extremely low numbers (~ 130 oocysts). In the present study, 1.5 x 102, 1.0 x 103, or 1.0 x 104 C. parvum oocysts were spiked into 10 l of source or finish water followed by recovery using Envirochek HV sampling capsules. A subsample of the recovered oocysts was analyzed by immunofluorscence assay (IFA) using the approved EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. Another subsample was subjected to DNA-RNA extraction, followed by RT-PCR using primers directed to the gene encoding Cryspovirus capsid. The IFA method showed that recovery of C. parvum oocysts at the 1.0 x 103 or 1.0 x 104 spiking levels were between 47 - 70% in both source and finish water. While oocysts were recovered from finish water inoculated with 1.5 x 102 C. parvum oocysts (45% recovery), no oocysts were recovered from source water at this spiking dose. Cryspovirus RT-PCR appeared to offer greater sensitivity than IFA because C. parvum oocysts were detected using this molecular technique in the source and finish water concentrates at all 3 spiking levels. A linear relationship was observed between log oocysts spiking dose and the relative intensity of the Cryspovirus RT-PCR signal for finish water, but not for source water. These data indicate that Cryspovirus RT-PCR may be a more sensitive method to detect C. parvum oocysts in source and finish water even at extremely low oocyst levels.