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Giardiasis is a neglected parasitic disease that affects primarily children, in whom it delays physical and mental development. The pathophysiology of giardiasis in not well understood, and most reports have identified Giardia intestinalis trophozoites only in the lumen and on the brush border of the small intestine. We identified Giardia trophozoites within the epithelium of the small intestine of a lactose intolerance patient. The Giardia trophozoites were obtained and cultured in vitro. In addition, we demonstrated Giardia trophozoite invasion in an animal model. Giardia trophozoites invaded the intestinal mucosa and submucosa of infected gerbils. The invasive trophozoites were observed at 21, 30 and 60 days age, and the average numbers of invaded sites were 17 ± 5, 15 ± 4, and 9 ± 3, respectively. We found trophozoites between epithelial cells, at the base of empty goblet cells, in lacteal vessels and within the submucosa. The morphological integrity of the invasive trophozoites was demonstrated via electron microscopy. The analysis of the gerbils infected with the trophozoites of the WB reference strain did not show intraepithelial trophozoites. These results demonstrate another Giardia pathogenic mechanism, opening the door to numerous future studies.

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BACKGROUND: In Mexico giardiosis is the most prevalent parasitic disease in children. Treatment with antiparasitics derived from: nitroimidazoles, benzimidazoles and nitrofuranes have been used; but, some of them have undesirable side effects. Recently nitazoxanide (Ntz) was introduced in Mexico; however, there are few studies on Giardia duodenalis susceptibility to Ntz. OBJECTIVE: To determine G. duodenalis sensitivity to Ntz and compare it to tinidazole (Tnz). MATERIAL AND METHODS: Assays were performed in four G. duodenalis isolates: INP231087MM, INP210897-AXA1 obtained from humans infections, INP170693HG8, INP300693-CP5 from a cat and a dog, respectively. Half million trophozoites were exposed to different Ntz or Tnz concentrations in TYI-S-33, for 24 hours at 37 degrees C. Trophozoite viability was tested by the colorimetric method of MTT-tetrazolium salts reduction to MTT-formazan. To analyze ultrastructural damage, control and experimental samples were processed by standard electron microscopy methods. Experiments were carried out, in a double blind in triplicate and repeated four times. Results were analyzed by variance analysis. RESULTS: Susceptibility at 100% were in a Ntz concentration ranging from 1 microgram to 7 micrograms and in a Tnz concentration ranging from 1 microgram to 4 micrograms. There were significant differences when sensitivities to Ntz were compared between them (P < 0.001). The ultrastructural analysis showed changes in trophozoite volume, loss of characteristic shape and swelling. CONCLUSIONS: This is the first report of G. duodenalis susceptibility to Ntz performed in isolates from different hosts. G. duodenalis isolates were more susceptible to Tnz than Ntz.

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We report two patients, one symptomatic and another asymptomatic, infected with Cyclospora an enteropathogen recently described. The symptoms were: abdominal pain, hiporexia, vomit, bruxism, watery diarrhea 3 or 4 times in a day but self-limiting after 24 to 72 hours. In both children there were unsporulated oocysts with a diameter of 8.44 +/- 0.146 microns detected in feces concentrated by Faust's technique; the confirmation was achieved with a Ziehl-Neelsen stain and the observation of sporulated oocysts (two sporocysts per oocyst). In Mexico, Cyclospora is perhaps overlooked or it is mistaken with oocysts of Cryptosporidium.

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OBJECTIVE: To investigate antigiardiasic activity in plants used in Mexico as antidiarrheics and/or antiparasitics. MATERIALS AND METHODS: Fourteen species were evaluated. The antigiardiasic activity was measured in vitro in a blinded fashion using trophozoites of Giardia duodenalis incubated with plant extracts. The viability of trophozoites was ascertained using MTT (3-[4,5-dimethylthiazol-2-il]-2, 5-diphenyl tetrazolium bromide) which is reduced to MTT-formazan by the activity of live trophozoites. The reduced MTT was extracted with an acidified alcohol (2-propanol with HCI 0.04 M) and measured in a spectrophotometer at 570 nm. Negative (trophozoites without extract) and positive controls (incubated with tinadazol) were included. RESULTS: The scientific and trivial names of the plants are given (trivials in Spanish marked by an asterisk). They had the following trophozoite mortality (mean +/- SD in percent): Justicia spicigera (muicle*) = 91 +/- 0.5; Lipia beriandieri (oregano) = 90 +/- 0.6; Psidium guajava (guava) = 87 +/- 1.0; positive control of tinidazol = 79 +/- 1.9; Punica granutus (granado*) = 78 +/- 1.3; Magnifera indica (mango) = 77 +/- 1.0; Plantago major (lante*) = 76 +/- 1.2; Cupressus semperbirens (cipres) = 73 +/- 1.2; Castella tormentosa (chaparro amargoso*) = 70 +/- 0.7; Hematoxilon campechanum (palo de Campeche*) = 67 +/- 1.2. Without or with a low mean activity were Prosopis juliflora (mesquite*) and Rizophora mangle (mangle*) with 0%, Oriza sativa (rice) with 5%, Capsicum annum (pimiento*) with 21% and Persea americana (avocado) with 23%. There were no associations of the antigiardiasic effect with concentration or osmolality of the extracts. CONCLUSIONS: A clear in vitro antigiardiasic effects was seen in nine species. Three of them were superior to tinidazol which is a drug of common use in the treatment of giardiasis.

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We wish to propose a data base of the isoelectric point (pl) for Giardia duodenalis isozymes [corrected]. As a contribution to this end we characterized by electrophoresis the isozymes of the malic enzyme (ME) of ten G. duodenalis isolates from Mexican children. Isoelectric focusing was performed in a vertical system using a one mm slab gel of 5.5% acrylamide prepared with 6.5% carrier ampholytes, pH 3-10, and 10% glycerol. Each test sample (10 micrograms) and a series of protein markers of known pl were applied in duplicate, and a minimum of six samples for each isolate were prefocused at 1.5 W, 200 V for 0.25 hour and focused at 2100 volt-hour, 3 W, 800 V. With the regression equation for protein markers Y = 1.886 + 5.709(X) (standard error for X = 0.001 and Y = 0.136), we calculated the pls for each isozyme of the malic enzyme detected in the G. duodenalis isolates. The pl of the isozymes were between 5.70-7.63 and the clone INP-100588-CMG1 was different from the parental isolates in three isozymes: pl 7.34, 7.16 and 6.99. The determination of isoelectric points of the isozymes of other enzymes of Giardia duodenalis should be a useful tool for the detection of their genetic variability by numeric comparison.

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In this paper, we report the hatching of Giardia spp. trophozoites from purified cysts harvested in Sheather's solution from human feces. Isolated cysts were induced by acidic Hank's Balanced Salt Solution (HBSS), incubated at 37 degrees C for 30 minutes, concentrated by centrifugation and washed with pH 7.5 HBSS. The cysts were placed in 8 ml. culture medium (Pancreatic digest of casein and yeast extracts) supplemented with human serum and bile in borosilicate glass tubes with screw caps. Trophozoite excystation was scanned in a Commandon chamber filled with the suspension, sealed with a cover glass and vaseline. The trophozoite emergence was recorded with phase contrast in a photomicroscope. The excystation began within the first 3 minutes, after inoculation into medium. The trophozoites emerged trough a small hole at one end of the cyst and simultaneously began cellular division, the whole process required 5-30 minutes. The trophozoites attached themselves to the walls of the culture tube, permitting the replacement and removal of contaminants without dislodging them from the tube walls. The isolates MM-INP and MG-INP have been maintained at 37 degrees C for more than six months.

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