Label-free detection and quantification of Giardia duodenalis cysts using a lens-free microscope

Abstract

Giardiasis leads to severe diarrhea, and its impacts are especially concerning in low-income sites that lack water and sanitation infrastructure. Monitoring water quality considering the infective form of Giardia is therefore an action that strongly aligns with public health. The standard protocol for cyst quantification in water involves immunofluorescence assays (IFA), which depend on fluorescent probes coupled to antibodies, making it an observer-dependent, and a relatively expensive method. Lens-free holographic microscopy (LHM) offers a possible alternative for imaging, as it relies on portable, lightweight, and low-cost equipment. It is based on phase and amplitude images of holograms recorded with partially coherent lighting. This study, therefore, aimed to test LHM for the detection and counting of Giardia cysts. Purified suspensions of G. duodenalis cysts, with known concentration, were quantified by IFA and LHM. Holograms were acquired by transmission, using three different wavelengths, and were then submitted to automated analysis, using an algorithm developed in Python. Slide preparation and cyst counting by IFA proved to be more laborious compared to LHM. Agglomerations of cysts impaired both methods. LHM quantification showed significant similarity with the reference number of cysts in suspension, which was not obtained by standard immunofluorescence. LHM proved to be an effective technique for detecting and counting Giardia cysts in purified samples and with high concentrations, showing potential for future application in water quality assessment targeting pathogens, but limitations include low sensitivity and a demand for future work considering matrix effect on real environmental samples.