Toxoplasma gondii: Prolonged in-vitro maintenance of virulent tachyzoites in fluid media at low temperatures

Background: Prolonged maintenance of infective Toxoplasma gondii tachyzoites (T.g.T.) is an important subject for research purposes. This study aimed to evaluate four serum free fluid media for prolonged in vitro maintenance of T.g.T.Methods: The four fluid media Phosphate buffered saline (PBS) pH 7.2 and Roswell Park Memorial Institute (RPMI-1640) with or without 3% fetal bovine serum (FBS) were evaluated for maintenance of virulent T.g.T. The four media were tested after incubation at three different temperature degrees in the darkness.Results: Prolonged maintenance period for infective T.g.T. was recorded especially in the absence of FBS supplement. RPMI without FBS was able to maintain infective T.g.T. for 16 days post incubation (dpi) at refrigerator temperature. This period decreased to 10 dpi and 6 dpi after incubation in the same media at 18–22 oC and 37 oC, respectively. Cultivation of T.g.T. in RPMI supplemented with 3% FBS and in PBS proved to maintain infective T.g.T. for 14 dpi at refrigerator temperature, and for 9 and 5 dpi when the two media were incubated at 18–22 oC and 37 oC, respectively. Shorter periods for keeping the T.g.T. infectivity were recorded using PBS supplemented with 3% FBS under all tested temperature conditions.Conclusion: This method allows economic long-lasting maintenance of tachyzoites for 16th dpi in RBMI that can be reactivated by reinoculation in mice.Keywords:  Toxoplasma gondii, Tachyzoites, Fluid media, Temperatur

Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt

Acanthamoeba keratitis (AK) is a severe corneal infection that may occur as a serious outcome of improper use of contact lenses (CL). Objectives: The study aimed to diagnose AK in soft CL users presenting with infectious keratitis, and to identify the prevalent genotypes isolated from different cases. Another aim was to determine the CL hygiene-related risk behaviors, and to explore the risk of water exposure for developing AK. Methods: A cross sectional study was performed. A questionnaire was carried out including 260 clinically diagnosed cases as infectious keratitis (170 females and 90 males); all of them were soft CL users for the suspected risk factors. Corneal scrapes from the affected eyes were cultured to diagnose bacterial and AK. PCR was performed and the amplified products were sequenced and compared with GenBank data. Results: The parasite was positively amplified from 32 samples (12.3%). Acanthamoeba T4 genotype was identified in 27/32 (84.4%) of isolates. Other detected genotypes belonged to T5 and T3 genotypes at rates of 9.4%, and 6.25%, respectively. The most important risk factors associated with development of AK were female sex, sleeping while wearing CL, and exposure to water resources through different practices. These practices included rinsing the CL case in tap water, swimming and/or showering while wearing CL, using multipurpose solution for cleaning the lenses, using water from over-building tanks. Rubbing the eyes due to discomfort when applying CL was an additional important risk factor associated with AK. The protective factor was regular hand washing before using CL. Conclusion: CL users were more exposed to AK and should gain enough health education regarding proper lens hygiene and dangers of tap water exposure. Keywords: Acanthamoeba keratitis, Soft contact lens, Genotyping, Hygiene, Cleaning solutions, Tap wate

Invasion biology meets parasitology: a case study of parasite spill-back with Egyptian Fasciola gigantica in the invasive snail Pseudosuccinea columella.

The liver fluke Fasciola gigantica is a trematode parasite of ruminants and humans that occurs naturally in Africa and Asia. Cases of human fascioliasis, attributable at least in part to F. gigantica, are significantly increasing in the last decades. The introduced snail species Galba truncatula was already identified to be an important intermediate host for this parasite and the efficient invader Pseudosuccinea columella is another suspect in this case. Therefore, we investigated snails collected in irrigation canals in Fayoum governorate in Egypt for prevalence of trematodes with focus on P. columella and its role for the transmission of F. gigantica. Species were identified morphologically and by partial sequencing of the cytochrome oxidase subunit I gene (COI). Among all 689 snails found at the 21 sampling sites, P. columella was the most abundant snail with 296 individuals (42.96%) and it was also the most dominant species at 10 sites. It was not found at 8 sites. Molecular detection by PCR and sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) revealed infections with F. gigantica (3.38%), Echinostoma caproni (2.36%) and another echinostome (7.09%) that could not be identified further according to its sequence. No dependency of snail size and trematode infection was found. Both high abundance of P. columella in the Fayoum irrigation system and common infection with F. gigantica might be a case of parasite spill-back (increased prevalence in local final hosts due to highly susceptible introduced intermediate host species) from the introduced P. columella to the human population, explaining at least partly the observed increase of reported fascioliasis-cases in Egypt. Eichhornia crassipes, the invasive water hyacinth, which covers huge areas of the irrigation canals, offers safe refuges for the amphibious P. columella during molluscicide application. As a consequence, this snail dominates snail communities and efficiently transmits F. gigantica

Total numbers of the snail species found at all sites.

<p>Total numbers of the snail species found at all sites.</p

Number of infected <i>P. columella</i> and prevalences for each trematode and site (total no. of tested snails given in brackets).

<p>Results sorted in decreasing order by prevalence of all trematodes.</p

Number and species of snails collected at the different sites. Sorted by number of indidiviuals per site.

<p>Number and species of snails collected at the different sites. Sorted by number of indidiviuals per site.</p

Total prevalence of the trematodes detected in <i>P. columella</i>.

<p>Error bars: 95% CI.</p

Sequences of primers designed for the present study and annealing temperatures.

<p>Sequences of primers designed for the present study and annealing temperatures.</p