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

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.

Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water.

AIMS: To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water. METHODS AND RESULTS: The method incorporated concentration of water samples by Al(2)(SO(4))(3)-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples. CONCLUSIONS: The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.

Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria.

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.