Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining

ABSTRACT

Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization (α = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither live G. muris cysts or C. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.

GRAPHICAL ABSTRACT

KEYWORDS:

• Viability assessment
• pathogenic protozoa
• propidium iodide
• fluorescent dyes
• cyst staining

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The São Paulo Research Foundation (FAPESP) [grant number 12/50522-0] and the Global Challenges Research Fund (GCRF) UK Research and Innovation [SAFEWATER; EPSRC Grant Reference EP/P032427/1] supported this work. The Coordination for the Improvement of Higher Education Personnel [CAPES-PROEX – Financial code 001] granted Kamila Jessie Sammarro Silva with a Master’s scholarship.