Use of the PEG/phosphate aqueous two-phase system in the separation of Cystoisospora belli: A new diagnostic method

Cystoisospora belli is an opportunistic apicomplexan parasite that infects the human gastrointestinal tract and causes cystoisosporiasis, particularly affecting individuals in tropical and subtropical regions and immunocompromised patients. Conventional parasitological diagnosis, although widely used due to its low cost, presents limitations such as low sensitivity and false-negative results. This study evaluated the potential of an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and phosphate for the separation and diagnosis of C. belli oocysts. Twenty assays were conducted based on a full factorial design (2⁴), considering PEG molecular weight, PEG and phosphate concentrations, and pH. The system produced three distinct phases: a PEG-rich top phase, an interface containing lipids and fecal debris, and a phosphate-rich bottom phase. Micromorphological analysis demonstrated that C. belli oocysts preferentially partitioned into the PEG-rich top phase, while the interface retained fecal residues and the bottom phase remained free of oocysts. The method generated slides with high clarity and minimal contamination, enabling clear visualization of oocyst morphology without structural damage. The PEG molecular weight influenced separation performance, with PEG 4000 combined with phosphate at pH 6, both at 10% concentrations, showing the best results. The most efficient system provided clear oocyst recovery in the polymer-rich phase with minimal residual contamination and required only 2 minutes for phase separation. Compared with conventional parasitological techniques, the ATPS approach demonstrated advantages such as faster processing, reduced use of toxic reagents, and improved sample clarification.