This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.The genus Entamoeba comprises six species (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba poleki, Entamoeba coli, and Entamoeba hartmanni) that live in the human intestinal lumen. E. histolytica, E. dispar, and E. moshkovskii are morphologically identical but are different biochemically and genetically (1,2,3,5,6). Although E. histolytica is recognized as a pathogen, the ability of the other two species to cause disease is unclear. E. moshkovskii, for example, is considered primarily a free-living ubiquitous amoeba found in anoxic sediments (2), and E. dispar is considered primarily a commensal of the human gut (3,5).Early studies of amebiasis in Australia have reported that the incidence of Entamoeba species varies from 1 to 4% in urban and rural communities, respectively (14). In another study, Law et al. (8) reported a 37% prevalence of Entamoeba in men who have sex with men. However, these studies did not differentiate E. histolytica from E. dispar or E. moshkovskii. The prevalence of E. histolytica, E. dispar, and E. moshkovskii (hereafter called the E complex) in the Australian population therefore remains unknown. The present study investigated the presence of the E complex in clinical samples by microscopy and PCR directly in stool samples collected from patients presenting with gastrointestinal symptoms.All of the stool specimens (from a diverse patient population) submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, Australia, during January 2003 to June 2006 for investigation of diarrhea were included in this study. Specimens from outpatients were collected by the patient and submitted to the laboratory as fresh specimens along with a portion mixed with sodium acetate-acetic acid-formalin (SAF). Specimens from inpatients or received without a portion fixed in SAF were immediately preserved in SAF upon arrival at the laboratory. The SAF-fixed specimens underwent permanent staining with a modified iron-hematoxylin stain according to the manufacturer's recommendations (Fronine, Australia). Patients diagnosed with members of the E complex by microscopy of stained smears underwent further investigations.DNA was extracted from fecal specimens (without fixatives) from patients either fresh or after storage at Ϫ20°C (immediately frozen upon arrival at the laboratory). Briefly, the stool sample (200 mg) was washed twice with 1 ml of sterile phosphate-buffered saline (pH 7.2), centrifuged for 5 min at 14,000 ϫ g, and DNA extracted according t...