Laboratory methods to distinguish between E histolytica and E dispar

Isoezyme analysis

By starch gel or polyacrylamide gel electrophoresis of cultures of E. histolytica/dispar from stools it is possible to distinguish between pathogenic and non-pathogenic strains using electrophoretic banding patterns of four enzymes: hexokinase, malic enzyme, glucosephosphate isomerase, and phosphoglucomutase. These methods have been used to distinguish Entamoeba isolates into 18 zymodemes, of which zymodeme II seems to be a marker for invasive E. histolytica 'in the Western world' (Healy 1988).

PCR to detect E. histolytica

DNA hybridization technology is capable of distinguishing between E. histolytica and E. dispar (Clark and Diamond 1991; Clark 1993) and the methods have evolved to become important tools in the demonstration of E. histolytica in clinical samples. A number of PCR primers designed to detect E. histolytica have been reported. Sequence analysis of homologous cDNA clones derived from pathogenic and non-pathogenic isolates of E. histolytica revealed 10% nucleic acid substitutions (Tannic and Burchard, 1991). Oligonucleotide primers specific for the gene encoding the 30-kDa molecule of pathogenic E. histolytica detected the parasite in 19 liver abscess fluids from 14 patients with a presumptive amoebic liver abscess. Only 2 of the 19 samples were positive microscopically (Tachibana et al. 1992). Primers detecting E. histolytica actin gene (Huber et al. 1988) detected the parasite in 22 out of 23 suspected liver abscess cases from Nicaragua (Linder et al. 1997).

Detection of E. histolytica-specfic antigens

Immunofluorescence can detect E. histolytica/dispar trophozoites and cysts with high sensitivity, but antibody reagents specific for E. histolytica cysts have been difficult to achieve (see, e.g.

Page 100

Perez et al. 1987), and despite encouraging reports in the literature on the detection of cysts in stool samples using monoclonal antibodies as markers, no such reagents are currently available commercially. An ELISA that detects the Gal/GalNAc lectin antigen and can distinguish between E. histolytica and E. dispar has been introduced commercially (Petri 1996). It appears to be useful, but its use is restricted to fresh stool samples. The target antigen appears to be rapidly destroyed even in frozen samples.

0 0

Post a comment