Summary: Blastocystis is an unusual enteric protozoan parasite of humans and many animals. It has a worldwide distribution and is often the most commonly isolated organism in parasitological surveys. The parasite has been described since the early 1900s, but only in the last decade or so have there been significant advances in our understanding of Blastocystis biology. However, the pleomorphic nature of the parasite and the lack of standardization in techniques have led to confusion and, in some cases, misinterpretation of data. This has hindered laboratory diagnosis and efforts to understand its mode of reproduction, life cycle, prevalence, and pathogenesis. Accumulating epidemiological, in vivo, and in vitro data strongly suggest that Blastocystis is a pathogen. Many genotypes exist in nature, and recent observations indicate that humans are, in reality, hosts to numerous zoonotic genotypes. Such genetic diversity has led to a suggestion that previously conflicting observations on the pathogenesis of Blastocystis are due to pathogenic and nonpathogenic genotypes. Recent epidemiological, animal infection, and in vitro host-Blastocystis interaction studies suggest that this may indeed be the case. This review focuses on such recent advances and also provides updates on laboratory and clinical aspects of Blastocystis spp.

New insights on classification, identification, and clinical relevance of Blastocystis spp.

The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease.

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Laboratory Diagnostic Techniques for Entamoeba Species

The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples.

Laboratory Diagnosis of Amebiasis

The recent identification of Entamoeba dispar as a separate species, which is nonpathogenic for humans but morphologically indistinguishable 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 Hue (TT Hue) in central Vietnam, a region known for its high incidence of invasive amebiasis. In addition, in a particular area of Hue 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 Hue 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.

Epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central Vietnam.

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.

Real-Time PCR for Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar in Fecal Samples

The antibody patterns--obtained by enzyme immunoassay (EIA), complement fixation (CF), indirect hemagglutination (IHA), and latex agglutination (LA)--of 216 patients with amebic liver abscess (ALA) were related to the time of onset of illness and to epidemiological data. The usual period between the stay in an Entamoeba histolytica-endemic area and clinical manifestation of the disease was 8-20 weeks. Increasing antibody concentration affected the sensitivity rates for CF and LA in the first 3 weeks of clinical ALA. In comparison with CF, IHA, and LA, EIA was the most sensitive test, showed the most substantial changes in antibody concentration according to the stage of disease, and measured persisting antibodies for the longest period. Neither the duration of previous stay in tropical countries nor the time when specific chemotherapy was started influenced development or persistence of antibodies in ALA patients. ALA occurred with similar frequency regardless of whether the patient was a long-term resident of the tropics or a short-term visitor to an E. histolytica-endemic area.

Development and persistence of antibodies to Entamoeba histolytica in patients with amebic liver abscess. Analysis of 216 cases.

Results are reported of antibodies to E. histolytica antigen obtained by enzyme immunoassay, complement fixation, indirect hemagglutination and latex agglutination in sera of persons with varying exposure to infection by E. histolytica. The data are compared to findings in non-exposed persons and in patients with amebic liver abscess. From the results it is concluded that the value of serodiagnosis in amebiasis is higher in patients, who have traveled in tropical areas for a limited period, than in residents of the tropics.

Efficiency of serodiagnosis in amebiasis. Results obtained by four different tests in 13 458 persons with varying exposure.

Serum samples of 107 patients with cystic and 30 with alveolar hydatid disease were tested for antibodies with hydatid fluid and extract from cyst material of E. alveolaris as antigens. Complement-fixation, indirect haemagglutination and an indirect enzyme immune (ELISA) technique were employed. The results indicate an antibody picture which confirms the value of serum-antibodies in the diagnosis of hydatid disease. Diagnostically insufficient antibody formation was present in seven patients with cystic and two with alveolar hydatid disease. The indirect haemagglutination test with alveolaris antigen proved to be the most sensitive reaction for antibody demonstration of both echinococci, while the same antigen in the ELISA technique gave species-specific results in patients with E. alveolaris.

[Serum antibodies in cystic and alveolar hydatid disease (author's transl)].

Determination of malarial antibodies by means of screening and differentiating antigens

Tests for serum antibodies against two amoeba antigens were performed on serum samples from 70 patients with amoebic liver abscesses, 204 travellers returning from tropical countries and 136 inhabitants of an endemic region without evidence of amoebiasis. Suspicious or borderline results in the complement-fixation, indirect haemagglutination, latex-agglutination, and double-gel diffusion tests occurred in 73.3%-90.4% of patients with amoebic liver abscess, in the same reactions (except double-gel diffusion test) in 12.1-18.9% of travellers in the trophics, and 16.2%-46.3% of inhabitants of endemic regions. There was evidence for the need of performing several tests: patients with amoebic liver abscesses gave diagnostically useful results the more frequently the more tests were performed on a given serum sample. Negative results in the three tests occurred in only one case of multiple liver abscesses, but the sample had been obtained very early.

E. Mannweiler

Papers

Development and persistence of antibodies to Entamoeba histolytica in patients with amebic liver abscess. Analysis of 216 cases.

Efficiency of serodiagnosis in amebiasis. Results obtained by four different tests in 13 458 persons with varying exposure.

[Serum antibodies in cystic and alveolar hydatid disease (author's transl)].

Determination of malarial antibodies by means of screening and differentiating antigens

[Serum-antibodies in patients with extra-intestinal amoebiasis (amoebic liver abscess) and healthy persons (author's transl)].