In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets.

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.

Entamoeba chiangraiensis n. sp. (Amoebozoa: Entamoebidae) isolated from the gut of Asian swamp eel (Monopterus albus) in northern Thailand.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 µm in length and 9-13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.

Vermamoeba vermiformis as etiological agent of a painful ulcer close to the eye.

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Collaborative intelligence and gamification for on-line malaria species differentiation.

BACKGROUND: Current World Health Organization recommendations for the management of malaria include the need for a parasitological confirmation prior to triggering appropriate treatment. The use of rapid diagnostic tests (RDTs) for malaria has contributed to a better infection recognition and a more targeted treatment. Nevertheless, low-density infections and parasites that fail to produce HRP2 can cause false-negative RDT results. Microscopy has traditionally been the methodology most commonly used to quantify malaria and characterize the infecting species, but the wider use of this technique remains challenging, as it requires trained personnel and processing capacity. OBJECTIVE: In this study, the feasibility of an on-line system for remote malaria species identification and differentiation has been investigated by crowdsourcing the analysis of digitalized infected thin blood smears by non-expert observers using a mobile app. METHODS: An on-line videogame in which players learned how to differentiate the young trophozoite stage of the five Plasmodium species has been designed. Images were digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Images from infected red blood cells were cropped and puzzled into an on-line game. During the game, players had to decide the malaria species (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi) of the infected cells that were shown in the screen. After 2 months, each player's decisions were analysed individually and collectively. RESULTS: On-line volunteers playing the game made more than 500,000 assessments for species differentiation. Statistically, when the choice of several players was combined (n > 25), they were able to significantly discriminate Plasmodium species, reaching a level of accuracy of 99% for all species combinations, except for P. knowlesi (80%). Non-expert decisions on which Plasmodium species was shown in the screen were made in less than 3 s. CONCLUSION: These findings show that it is possible to train malaria-naïve non-experts to identify and differentiate malaria species in digitalized thin blood samples. Although the accuracy of a single player is not perfect, the combination of the responses of multiple casual gamers can achieve an accuracy that is within the range of the diagnostic accuracy made by a trained microscopist.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Giardia duodenalis assemblage-specific induction of apoptosis and tight junction disruption in human intestinal epithelial cells: effects of mixed infections.

In view of the interest in genotype-specific pathogenesis in Giardia duodenalis , the aim of the present study was to examine the effects of infection with different, or mixed, G. duodenalis assemblages on the integrity of human intestinal epithelia. To that end, human epithelial cells (HCT-8) were cultured and exposed to different G. duodenalis assemblages (A, B, and E) or a combination of these assemblages. Epithelial disruption and apoptosis were evaluated by fluorescent microscopy and apoptotic oligonucleosome quantification. The results indicate that infection with trophozoites disrupts epithelial tight junctions and induces varying degrees of enterocyte apoptosis, depending on the infecting assemblage. All disruptions were caspase-3 dependent and were more pronounced when caused by a non-host specific assemblage. Furthermore, infections by isolates in combination with isolates from another assemblage enhanced the epithelial disruption and apoptosis. Further studies in vitro and in vivo are required to confirm the mechanisms of enhanced pathogenicity of mixed or non-host specific (or both) G. duodenalis infections. Findings in the present study point to the potential pathogenic importance of intra-species polyparasitism in giardiasis.

Species boundaries in gregarine apicomplexan parasites: a case study-comparison of morphometric and molecular variability in Lecudina cf. tuzetae (Eugregarinorida, Lecudinidae).

Trophozoites of gregarine apicomplexans are large feeding cells with diverse morphologies that have played a prominent role in gregarine systematics. The range of variability in trophozoite shapes and sizes can be very high even within a single species depending on developmental stages and host environmental conditions; this makes the delimitation of different species of gregarines based on morphological criteria alone very difficult. Accordingly, comparisons of morphological variability and molecular variability in gregarines are necessary to provide a pragmatic framework for establishing species boundaries within this diverse and poorly understood group of parasites. We investigated the morphological and molecular variability present in the gregarine Lecudina cf. tuzetae from the intestines of Nereis vexillosa (Polychaeta) collected in two different locations in Canada. Three distinct morphotypes of trophozoites were identified and the small subunit (SSU) rDNA was sequenced either from multicell isolates of the same morphotype or from single cells. The aim of this investigation was to determine whether the different morphotypes and localities reflected phylogenetic relatedness as inferred from the SSU rDNA sequence data. Phylogenetic analyses of the SSU rDNA demonstrated that the new sequences did not cluster according to morphotype or locality and instead were intermingled within a strongly supported clade. A comparison of 1,657 bp from 45 new sequences demonstrated divergences between 0% and 3.9%. These data suggest that it is necessary to acquire both morphological and molecular data in order to effectively delimit the "clouds" of variation associated with each gregarine species and to unambiguously reidentify these species in the future.

Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites.

Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.