Label-Free Darkfield-Based Technique to Assist in the Detection of Giardia Cysts

Author(s)

Belini, Valdinei Luis ^[1] ; Souza Freitas, Barbara Luiza ^[2] ; Sabogal-Paz, Lyda Patricia ^[2] ; Branco, Nilson ^[3] ; Bueno Franco, Regina Maura ^[3]

Affiliation

^[1] Univ Fed Sao Carlos, Dept Elect Engn, RodoviaWashington Luis, Km 235, BR-13565905 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Sao Carlos Sch Engn, Dept Hydraul & Sanitat, Trabalhador Sao Carlense Ave 400, BR-13566590 Sao Paulo - Brazil

^[3] Univ Estadual Campinas, Inst Biol, Protozool Lab, BR-13083970 Campinas, SP - Brazil

• Document type: Journal article
• Source: WATER AIR AND SOIL POLLUTION; v. 229, n. 6 JUN 2018.
• Web of Science Citations: 1

Abstract

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol's iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4',6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated. (AU)

• FAPESP's process: 12/50522-0 - Environmental contamination by protozoa Giardia spp. and Cryptosporidium spp. and Ascaris suum: challenges of detection, removal and inactivation of infective forms
• Grantee: Regina Maura Bueno Franco
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 14/12712-8 - BioSand filters operated in continuous and intermittent flows: challenges in the construction, operation and maintenance
• Grantee: Lyda Patricia Sabogal Paz
• Support Opportunities: Regular Research Grants