Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water

Abstract

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1 -L water replicates and concentrated using centrifugation, Al2(SO4)3 or Fe2(SO4)3 flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomal RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al2(SO4)3 flocculation. No PCR products were generated for samples that had been pre-treated using Fe2(SO4)3 flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

“…Prior to this study, several methods had been described for the recovery and detection of T. gondii oocysts in contaminated water (Isaac-Renton et al 1998, Dumètre & Dardé 2003, Kourenti & Karanis 2004, Sotiriadou & Karanis 2008. These methods were not as well established as the ones described for the recovery and detection of Cryptosporidium and Giardia.…”

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

“…Previously, the performance of conventional or nested PCR in the detection of T. gondii oocysts was evaluated in several studies, and the detection limits varied greatly between assays and studies. The sensitivity of an 18S rRNA nested PCR was reported to be 0.1 oocyst for distilled water (35), 100 oocysts/sample for river water, and 10 oocysts/sample for well and sea water (36). Similarly, the sensitivity of a B1-based TaqMan end point assay was more than 10 oocysts/liter for drinking water and more than 1,000 oocysts/liter for raw surface water (55).…”

“…PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32,35,36,46,49,55) over the conventional mouse bioassay (27,55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).…”

“…The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35,36,49,55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor.…”

“…Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16,35,36,46,55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30,55,57).…”