Use of polymerase chain reaction and nonradioactive DNA probes to diagnose Entamoeba histolytica in clinical samples

Abstract

E. histolytica parasites in Mexican children's stools were identified and typed as pathogenic or non-pathogenic using the polymerase chain reaction (PCR) and nonradioactive probes. PCRs were performed with primers specific for 145 base pair (bp) pathogenic or 133 bp non-pathogenic DNA sequences, which are highly repeated in E. histolytica parasites with pathogenic or non-pathogenic isoenzyme patterns, respectively. Dot-blotted PCR products were identified with a horseradish peroxidase-conjugated oligonucleotide probe specific for either the 145 bp pathogenic or 133 bp non-pathogenic sequences. The PCR and the 145 bp pathogenic probe correctly identified eight cultures with pathogenic isoenzyme types and none of nine cultures with non-pathogenic isoenzyme. The PCR and 133 bp non-pathogenic probe identified all of the non-pathogenic cultures, none of the axenized pathogenic cultures, and three of five xenic cultures with pathogenic isoenzymes. The two probes together identified all 49 stools containing E. histolytica by light microscopy (sensitivity = 1.0), which represented the entire set of the E. histolytica-positive stools diagnosed at the Hospital Infantil over a 10 week period. Most patient isolates were positive with both 145 bp pathogenic and 133 bp non-pathogenic probes, suggesting that these children, 60% of whom were dysenteric, are infected with mixed populations of amebas.