Abstract. The recent identification of Entamoeba dispar as a separate species, which is nonpathogenic for humans but morphologically indistinghuishable from Entamoeba histolytica, has prompted the World Health Organization to recommend reinforced efforts for reassessment of the epidemiology of amebiasis and, in particular, of E. histolytica. In this regard, the distribution of amebic liver abscess (ALA) cases were analyzed in the province of Thua Thien Hué (TT Hué) in central Vietnam, a region known for its high incidence of invasive amebiasis. In addition, in a particular area of Hué City, a parasitologic and seroepidemiologic survey was performed to identify possible risk factors for transmission of E. histolytica. Based on the analysis of hospital charts from April 1990 to April 1998, 2,031 cases of ALA were identified, indicating an ALA incidence of at least 21 per 100 000 inhabitants per year. Incidence varied substantially between the various districts of TT Hué and directly correlated with population density. The risk for ALA was significantly higher in summer and was age and sex dependent because 95% of the cases were adults, of which more than 80% were males. There was no clustering of cases within households and recurrent cases of ALA occured more frequently than predicted in the study population. Despite the higher incidence of ALA in males, the parasitologic and seroepidemiologic survey revealed a significant higher infection rate for intestinal protozoon parasites, including E. histolytica in females. Besides level of education and access to a toilet or tapwater, use of river water was identified as an important risk factor for E. histolytica infection.

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica-or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.

To gain insight into the dynamics of intestinal Entamoeba histolytica infection, a longitudinal study was performed over an observation period of 15 months with a group of 383 randomly selected adult individuals (mean age, 38.5 years) living in an area of amebiasis endemicity in central Vietnam. Ameba infection was diagnosed by using species-specific PCR and DNA extracted directly from fecal samples. The results indicated an E. histolytica prevalence of 11.2% and an annual new infection rate of 4.1% in the study population. Follow-up of the 43 individuals who were E. histolytica positive at enrollment suggested a regular exponential decline in infection of about 3% per month and a mean half-life of infection of more than 15 months. However, the reinfection rate for this group of participants was 2.7 times higher than that predicted for the study population as a whole. Both the reappearance of the parasite after successful treatment of E. histolytica infection and changes in "genetic fingerprints" of parasites during the course of infection revealed an annual new infection rate of about 11.5%. Thus, the mean half-life of E. histolytica infection was calculated to be 12.9 months (95% confidence interval, 10.2 to 15.6 months). Notably, none of the participants developed symptoms compatible with invasive intestinal amebiasis, and only one of the subjects developed an amebic liver abscess during the observation period.

Sequences of small-subunit rRNA genes have been obtained for four new isolates of Entamoeba. Phylogenetic analyses give new insights into the evolution of these organisms. A novel Entamoeba from pigs in Vietnam that produces uninucleate cysts proved to be unrelated to other uninucleated cyst-producing species. Revival of the name Entamoeba suis for this organism is proposed. Instead of being related to Entamoeba polecki, it shares a recent common ancestor with the non-encysting Entamoeba gingivalis in a lineage that is basal to the tetranucleate cystproducing clade. This suggests that species producing cysts with four nuclei are descended from an ancestor that produced cysts with a single nucleus. An Entamoeba from a horse was isolated in culture. No cysts were observed in the original stool sample but the sequence is placed unequivocally within the clade of tetranucleate cyst-producing species with no other sequences being specifically related. Revival of the name Entamoeba equi for this organism is proposed. The Entamoeba ecuadoriensis sequence was found to be the most closely related to Entamoeba histolytica and Entamoeba dispar, as predicted, despite the organism having been an environmental isolate originally assigned to Entamoeba moshkovskii. Finally, a partial E. polecki gene sequence from a pig proved to be virtually identical to that of Entamoeba struthionis from an ostrich, suggesting that the latter name is a synonym.

SummaryThirty-nine patients with amoebic liver abscess (ALA), admitted to the Central Hospital of Hué (Vietnam), were evaluated in a comparative, prospective and randomized study for the treatment of ALA. Adult patients with an abscess located in the right liver lobe and an abscess diameter of 6 to 10 cm were included. Bacterial abscesses were excluded by microbiological examination of abscess fluid in all patients. Nineteen patients were treated with metronidazole for 10 days alone and 20 patients were punctured under ultrasound guidance with aspiration of abscess fluid in addition to drug administration. The clinical symptoms fever, pain in right upper abdomen and liver tenderness, and the laboratory parameters erythrocyte sedimentation rate, white blood cells, haemoglobin and C-reactive protein and the abscess size were determined on the day of admission and followed during an observation period of 38 days. Improvement of liver tenderness was significantly faster in the aspiration group during the first 3 days (P < 0.001), whereas all the other parameters showed no differences between the two groups. This minor benefit is obviously not sufficient to justify routine needle aspiration and advocates drug treatment alone for uncomplicated amoebic liver abscesses with a diameter up to 10 cm located in the right liver lobe.keywords amoebic liver abscess, treatment, metronidazole, abscess puncture, Entamoeba histolytica, Vietnam