Table 1.
RT-PCR primers used in this work.
Figure 1.
Ultrastructural analysis of E. histolytica CLS resistant to detergents.
(A) Light microscopy of a CLS stained with Lugol's Iodine showing its spherical shape, multi-nucleation and partial refringence; (B, C and D) Transmission electron microscopy of CLS showing two, three and four nuclei, respectively (arrow heads). High number of cytoplasmic vacuoles are also observed; (E) and (F) Scanning electron microscopy showing the smooth surface, spherical shape and small size of the CLS. Inbox in F shows a magnification of a hole in the surface of a CLS, where overlapping fibers that resembles chitin-fibers in other organisms are observed [6],[7].
Table 2.
Detergent-resistance percentage (conversion rate) of E. histolytica trophozoites treated with treatment solution at different times.
Figure 2.
Detection of chitin in E. histolytica CLS resistant to detergents by calcofluor white staining (upper panel) and a WGA-binding assay (bottom graphic).
(A and B) Light and UV microscopy, respectively, of a mix culture of untreated trophozoites and CLS stained with calcofluor white. CLS is clearly differentiated from trophozoites by the spherical shape and small size. In B, blue fluorescence of CLS under UV contrasts with the absence of fluorescence in the trophozoites 20X; (C and D) Comparison of calcofluor staining of CLS (C) against an E. histolytica cyst isolated from human feces (D). In spite of both showing a similar blue-whitish fluorescence, staining of the surface is clearer in the cyst from human feces, whereas staining of some apparently internal structures (arrowheads), probably transporting vacuoles, is evident in the CLS (40X). In the bottom graphic, bars represent the standard deviations.
Figure 3.
Gln6Pi enzyme mRNA expression levels assessed by RT-PCR in E. histolytica CLS resistant to detergents.
Total RNA was purified from untreated trophozoites (lane a), cysts isolated from human feces (lane b) and the CLS (lane c) and RT-PCR amplified by using specific oligonucleotides for E. histolytica Gln6Pi gene. Clear over-expression of Gln6Pi is observed in CLS (25 folds) respect to the basal expression in untreated trophozoites. Parallel RT-PCR amplification of the ADP-ribosylating factor (ARF) for each sample was used as internal loading control.