PCR Detection of Entamoeba histolytica , Entamoeba dispar , and Entamoeba moshkovskii in Stool Samples from Sydney, Australia

Abstract

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...

Invasive Amebiasis in Men Who Have Sex with Men, Australia

“…Microscopic examination of permanently fi xed, stained fecal smears was positive for E. histolytica/dispar/moshkovskii complex. Diagnosis of E. histolytica was confi rmed by PCR targeting the small subunit ribosomal DNA as described (8). Results of serologic examination of these patients were positive; all titers were >256 according to indirect hemagglutination antibody assay, which confi rmed invasive disease.…”

Development of Multiplex Real-Time Polymerase Chain Reaction for Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in Clinical Specimens

“…1 This procedure is cheap and simple, but it has several limitations, such as being incapable of distinguishing between the cysts and trophozoites of the disease-causing species Entamoeba histolytica , the nonpathogenic Entamoeba dispar , and the free-living amoeba Entamoeba moshkovskii . A high prevalence and association of E. moshkovskii with E. histolytica and E. dispar has been reported in young children from Bangladesh 2,3 and E. moshkovskii was recently detected in patients between 31 and 50 years of age presenting with gastrointestinal symptoms in Australia 4 ; therefore, humans may be a true host for this amoeba. This observation has made the differentiation of the three species in clinical samples of great importance, both for diagnosis and for epidemiological studies.…”

“…6,8 However, most studies that have investigated the prevalence of E. histolytica and E. dispar have not considered the possible presence of E. moshkovskii , due partly to a lack of tools to detect E. moshkovskii other than cultivation, 12 which is laborintensive, not always successful, and problematic in the case of mixed infections. Recently, conventional PCR assays for E. moshkovskii in clinical diagnosis of amoebiasis have been performed 3,4,13,14 but most of the protocols reported so far require further processing of the amplicon, which is timeconsuming and prone to false-positive results caused by possible cross-contamination.…”

Seroprevalence of Entamoeba histolytica Infection among Men Who Have Sex with Men in Sydney, Australia

“…15 A study from Sydney showed high rates of intestinal parasites in MSM, with 52.2% of stool specimens positive for protozoa, compared with 13% from non-MSM, of which E. histolytica had a prevalence rate of 0.24% in the MSM group. 16 Another report of five cases of invasive amebiasis in MSM from Sydney further documents its existence in this at risk group.…”

“…4 Improved methods of detection, with greater sensitivity and specificity include antigen and antibody detection and polymerase chain reaction. 15,18 Serum antibody detection is most useful in patients with extra-intestinal disease and is able to detect antibodies specific for E. histolytica. Sensitivity of these tests is ~95% for patients with amoebic abscesses, 70-85% for patients with active amoebic colitis, and 10-20% for asymptomatic people passing cysts, with specificity of 95% generally reported, 19,20 and E. histolytica -specific antibodies persisting for many years after exposure.…”