SIMPLE DIFFERENTIAL DETECTION OF ENTAMOEBA-HISTOLYTICA AND ENTAMOEBA DISPAR IN FRESH STOOL SPECIMENS BY SODIUM-ACETATE ACETIC-ACID FORMALINCONCENTRATION AND PCR

Amoebiasis is caused by two distinct species, a pathogenic form (Entam oeba histolytica) and a nonpathogenic form (Entamoeba dispar), which a re morphologically identical. Although the distinction between these t wo species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious proce dures for isolation of the DNA. We report here a simple PCR method sta rting with fresh stool specimen that allows for the sensitive and reli able distinction between E. histolytica and E. dispar. After initial c oncentration by the sodium acetate-acetic acid-formalin (SAF) method a nd digestion with proteinase K, a 0.88-kb sequence of the multicopy 16 S rRNA gene served as a target for PCR amplification, The method start ing with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial c oncentration step, However, storage in SAF fixative prior to testing r esulted in a decreased sensitivity within 2 days, The detection limit of the method was as low as one copy of the 16S rRNA gene, No cross-re activity was observed with other common intestinal protozoa. Mixed inf ections involving both E. histolytica and E. dispar could easily be de tected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.