Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl-Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

The effects of Entamoeba histolytica lysates on human colonic mucins.

Detergent lysates of Entamoeba histolytica trophozoites contained high levels of beta-N acetyl-D-glucosaminidase, beta-N acetyl-D-galactosaminidase and alpha-D-galactosidase activity, and lower but significant levels of five other glycosidases. Although these activities should have been capable of largely degrading the oligosaccharide side-chains of human colonic mucin, in fact only about one third of high MW mucin was degraded in 72 h and trypsin alone produced a similar effect. There was no evidence that these glycosidases were excreted and we conclude that they are unlikely to represent significant virulence factors for E. histolytica.

Activity of disulfiram (bis(diethylthiocarbamoyl)disulphide) and ditiocarb (diethyldithiocarbamate) against metronidazole-sensitive and -resistant Trichomonas vaginalis and Tritrichomonas foetus.

Clinical resistance of Trichomonas vaginalis to metronidazole is best correlated with MIC values measured under aerobic conditions. Under these conditions both disulfiram (bis(diethylthiocarbamoyl)disulphide), and its first mammalian metabolite, ditiocarb (diethyldithiocarbamate), showed high levels of activity against metronidazole-sensitive (disulfiram MIC, 0.1-0.7 microM; ditiocarb MIC, 0.3-9 microM) and -resistant (MICs 0.2-1.3 microM and 1.2-9 microM respectively) isolates. Tritrichomonas foetus was also sensitive-the MICs for seven metronidazole-sensitive isolates were 0.1-1.0 microM for disulfiram and 1.0-6.9 microM for ditiocarb; those for two highly metronidazole-resistant strains were 0.3-1.3 microM and 0.6-6 microM respectively. Under anerobic conditions most strains became highly resistant to both compounds. Surprisingly, disulfiram was consistently more active than ditiocarb.

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.

Gut Coccidia--Isospora, Cryptosporidium, Cyclospora and Sarcocystis.

The gut Coccidia are members of a large, varied, and exclusively intracellular group of protozoan parasites, four species of which (Isospora, Cryptosporidium, Cyclospora, and Sarcocystis) are human pathogens. The first three, but particularly Cryptosporidium parvum, have moved from medical curiosities to major problems with the coming of the acquired immunodeficiency syndrome (AIDS) epidemic, but are now known to also cause disease in the immunocompetent patient. They are easy to acquire and difficult to remove from the environment and, in the case of cryptosporidiosis, impossible to treat properly. Further research into many aspects of the biology of these organisms is urgently needed.

A novel transcribed repeat element from Entamoeba histolytica.

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Field evaluation of an improved Kato-Katz thick smear technique for quantitative determination of helminth eggs in faeces.

A new method for the quantification of helminth eggs in faeces was developed, in which 7.5% nigrosin in 10% formaldehyde mixed with 5% eosin yellow in 10% formaldehyde was substituted for the malachite green solution used in the standard Kato-Katz method. This modification revealed the eggs of parasites like Schistosoma mansoni, Ascaris lumbricoides, Trichuris trichiura and hookworms distinctly. The slides made with this new technique could be accurately read within one hour. Faecal smears from 100 pupils in Kigungu, Entebbe, Uganda, were studied with both methods. The egg counts of S. mansoni, A. lumbricoides and T. trichiura by both methods were equal. The modified method, however, showed significantly higher hookworm egg counts (p < 0.001). Hookworm eggs were equal one hour after preparation of the slides as 16 hours after preparation. The intensity of infection detected was higher with the modified method for both S. mansoni and hookworms.

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5' and 3' regions.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.

ELISA for detection of anti-Giardia specific IgM: response in serum.

Infection with Giardia lamblia (G. duodenalis, G. intestinalis) is common all over the world, especially in children. Traditional diagnosis by faecal microscopy has only moderate sensitivity; serological tests, although not always positive, are acceptable to patients and useful in epidemiological studies. We show here that serum IgM separated by column chromatography and assayed by an indirect ELISA test can be a useful tool for the diagnosis of giardiasis. One hundred and thirty-nine positive sera (based on a single faecal examination), and 97 negative serum samples from Riyadh, Saudi Arabia, were examined. Taking positive results as being 2 s.d. above the mean of the controls, there were 117 positive results among the microscopically negative controls (3% false positives). The sensitivity of the test was 84% and the specificity 97%; the predictive value of a positive result is 97.5% and of a negative one 81%.

Influence of bacteria on electrophoretic proteinase patterns of Entamoeba histolytica isolates.

Gelatin SDS- polyacrylamide gel electrophoresis was used to compare proteinase banding patterns under reducing conditions from whole cell lysates of four axenic and four xenic pathogenic strains of Entamoeba histolytica. All strains shared major bands in the 34 and 66-68 kDa regions, whereas only the axenic strains produced major bands at 26, 28, 30 and 45 kDa. One axenic strain, NIH 200, when reassociated with mixed bacterial flora, reverted to an electrophoretic banding pattern characteristic of other xenic strains. These results suggest that the 26-30 kDa and 45 kDa proteinases of E. histolytica are induced by bacterial starvation while others are constitutively expressed. It is also proposed that the axenic bands of 45 and 34 kDa represent respectively, the reduced forms of the 56 and 40 kDa bands reported elsewhere under non-reducing conditions.

Enzyme-linked immunosorbent assay for the detection of anti-Giardia specific immunoglobulin G in filter paper blood samples.

Conventional diagnosis of infection with Giardia duodenalis is by faecal examination but the sensitivity of a single examination is low. Serological tests, although not always positive, are more acceptable to patients and are useful in determining the prevalence of giardiasis in large populations. We show here that blood collected on filter paper and dried provides an excellent source of material for enzyme-linked immunosorbent assays; using samples from a group of 88 stool-negative and 45 positive patients, the optimum results were obtained by taking the control mean optical density plus one standard deviation as the negative/positive cut-off value. The sensitivity was 91% (2/45 false negatives), and the specificity 95% (4/88 false positives). This method should be particularly useful for large-scale surveys in developing countries or wherever serological testing is done in central laboratories.