Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes.

We developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany.

Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2-5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400-6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4',6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0-56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey.

A total of 70 water samples, including tap, river, fountain and well water were collected in the Ordu province, Middle Black Sea, Turkey and investigated for the detection of Cryptosporidium oocysts. The samples were directly screened microscopically for Cryptosporidium oocysts' detection by immunofluorescence test and subsequently DNA was extracted for the molecular detection by loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Eighteen out of the 70 (25·7%) water samples were found positive for Cryptosporidium spp. by immunofluorescence test and 19 (27·1%) were found positive by LAMP. Nested PCR products were not generated in any of the investigated water samples. A total of 16 randomly selected pellets were spiked with 10 Cryptosporidium oocysts to test the efficiency of the applied method. All the samples were found positive by LAMP for the presence of Cryptosporidium DNA, while the nested PCR assay was positive in only seven (43·75%) out of the 16 examined spiked samples. This is the first report on the occurrence of Cryptosporidium species in environmental and drinking water supplies in the Black Sea area.

The potential of embryonic stem cells combined with -omics technologies as model systems for toxicology.

The derivation of pluripotent embryonic stem (ES) cell lines has opened up new areas of research in basic and applied science, most significantly in developmental biology and regenerative medicine. While application-oriented research has for the most part focussed on obtaining differentiated, organotypic cells from ES cells for future cell grafting therapies, ES cells have more immediate potential for use in toxicological in vitro assays used during drug development. ES cells are derived from blastocyst-stage embryos and offer an in vitro model for early development, thus enabling tests for teratogenicity testing in a human cell culture system and avoiding the pitfalls of inter-species differences. Differentiated, organotypic cells obtained from ES cells can potentially replace the primary cells and cell lines currently used for in vitro toxicology by offering a more consistent and potentially limitless source of differentiated cells. This can facilitate the establishment of comprehensive toxicogenomics and -proteomics databases and complement current databases that rely on data obtained from animal experiments. More recently, induced pluripotent stem (iPS) cells with ES cell-like properties have been obtained through reprogramming of somatic cells, thus enabling the generation of disease-specific cell lines. We review the potential of combining ES cells and ES cell-derived somatic cells with "omics" technologies for in vitro toxicology with a particular emphasis on the development of toxicogenomics and toxicoproteomics signatures. A separate section describes the potential of iPS cells for toxicology.

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.