SUPPLEMENTARY TOOLS FOR THE DIAGNOSIS OF AMOEBIASIS

SUPPLEMENTARY TOOLS FOR THE DIAGNOSIS OF AMOEBIASIS

Microscopic examination is a rather unreliable method for the correct identification of Entamoeba spp. in fecal specimens.  Even when carried out under optimal conditions (i.e. with permanent staining), sensitivity is never above 60%. Due to their morphological identity, Entamoeba histolytica, Entamoeba dispa, Entamoeba moshkovskii cannot be microscopically distinguished. Therefore, other tests are required to identify these species. In patients with dysentery, the presence of hematophagous trophozoites was for a long time considered sufficient for distinguishing Entamoeba histolytica from Entamoeba dispar. This criterion is no longer considered valid, because in some patients even Entamoeba dispar may phagocytize red blood cells;  this clinical observation has  been confirmed with in vitro experimentation.
Culture techniques to isolate Entamoeba spp. exist for several decades. Using this method, the success rate of Entamoeba histolytica identification varies among referral labs from 50% to 70%. Cultures undergo isoenzymatic typization by gel-electrophoresis (zymodemes) for species identification. This technique is used for research purposes only and not as a routine diagnostic procedure because of the long time needed for results (1-2 weeks).
Many tests have been developed (e.g. ELISA, immunochromatographic tests) to detect specific antigens of Entamoeba histolytica or Entamoeba dispar directly in fecal specimens.  Although a great deal of literature exists, data concerning sensitivity and specificity of these tests are contradictory (see “Bibliography”). The problem is that the results of studies on the accuracy of these tests are not always convincing due to the lack of a true reference method. One should also consider that the antigens detected by current methods (in some tests, the characteristics of the antigens are not even described) are specific for the trophozoitic membrane and are not present on the cystic wall.  However, Entamoeba histolytica/dispar cysts are almost always the only stage of the parasite that can be detected in feces of patients who are asymptomatic or free of diarrhea or dysentery. Furthermore, for a commercially available ELISA, cross-reactivity has been reported among Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii, a problem that obviously limits the usefulness of the test.
ELISAs are rapid (maximum, 3 h), simple and cheap and do not require skilled technicians, but one should know and consider their limits. Furthermore, their sensitivity and specificity should be improved. To date, no ELISA for the identification of Entamoeba moshkovskii is available. ELISAs carried out on the drainage liquid of amoebic liver abscesses have a sensitivity of 100% and a specificity of 90%-100% before drug treatment.
The introduction of molecular biology techniques, such as DNA extraction directly from stool sample and subsequent amplification of parasite-specific sequences by PCR, has provided new means to diagnose amoebiasis. These tests are at least 100-times more sensitive than ELISAs for the detection of antigens in fecal specimens.  Moreover, with nested multiplex PCR, it is possible to simultaneously detect the presence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii (trophozoites or cysts) in fecal specimens. However, the delivery or storage of specimens at room temperature leads to the rapid degeneration of DNA, especially in the vegetative forms of the parasite, which are the most fragile. Freezing the specimen at -20C (before DNA extraction) does not affect the sensitivity of the method.  Use of routine fixing agents for feces is currently under investigation. The main limitations of these tests are the high costs and the need for skilled technicians; the results are generally ready within 1-2 days. Many serological methods (i.e. search for antibodies) for the diagnosis of amoebiasis are available, the most common being indirect hemagglutination (IHA), latex agglutination, indirect immunofluorescence (IFA) and ELISA. Serological tests are specific but have different levels of sensitivity according to the clinical manifestations of the infection. In geographical areas where amoebiasis is endemic, these tests are unreliable because serum levels of anti-amoeba antibodies remain high for long periods (even years).  For these tests, the costs are low and the results are available, according to the method used, within 3 hours.

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