The application of modern laboratory techniques (PCR, ELISA, immunochromatographic tests) to the diagnosis of intestinal protozoa makes the process faster and more reliable in many cases. However, despite the undoubted value of these methods, stool microscopic examination remains the most reliable and economic tool to identify intestinal protozoa in most laboratories, due to limited financial resources and the small number of samples to be analyzed. Because of the limits of microscopic analysis, those who use this diagnostic method should have an appropriate training (see, for example, and should participate in continuing professional development (quality control). It is not surprising that parasitology is considered the lowest ranked branch of the analytical laboratory, receiving limited investments in both human and financial resources. A well-performed stool concentration technique is usually considered adequate for identifying most intestinal parasites and for detecting protozoan cysts and helminthic eggs and larvae. Unfortunately, this method makes it difficult or even impossible to identify vegetative forms of protozoa (trophozoites) that might be present in the sample: this difficulty may be due not only to the limited number of trophozoites in the sample but also to the considerable variability in shape and size within one species (these variations are also due to the stage of division of the vegetative form). One must also consider the possible presence in the same sample of multiple species with similar morphological features.

The results of a microscopic examination can also be compromised by the procedure with which the stool sample was collected: samples brought to the laboratory have often been collected several hours before or even the day before (in the latter case, the vegetative forms will probably be degraded) or have been collected in fixatives that preclude the use of permanent staining. Even with hospitalized patients, there can be difficulties of all kinds in obtaining appropriate samples. The use of permanent stains on fresh or correctly fixed samples facilitates the recognition of vegetative forms, but few laboratories routinely perform these procedures, due to lack of adequate training, laziness, or frustration in having to examine samples that are usually negative. Even though permanent staining methods improve the microscopic identification of many species of amoeba, the problem remains largely unresolved regarding the diagnosis of Entamoeba histolytica. It took 70 years for the scientific community to officially recognize the work of Emile Brumpt who, in 1925, hypothesized and demonstrated the existence of Entamoeba dispar, a non-pathogenic species microscopically indistinguishable from Entamoeba histolytica. Numerous studies have indicated that the prevalence of Entamoeba dispar is much greater than that of Entamoeba histolytica, and this has notably changed the epidemiology of amoebiasis worldwide. Microscopic examination, even of permanent stained samples, is insufficient for the differential diagnosis between Entamoeba histolytica and Entamoeba dispar or between Entamoeba histolytica and Entamoeba moshkovskii, morphologically identical species. In these cases, one should use particular techniques, such as stool culture followed by zymodeme analysis, PCR or ELISA performed directly on fecal specimens. However, currently available ELISAs should be improved in terms of sensitivity, specificity and the possibility of diagnosing Entamoeba moshkovskii because - even if data are limited - this amoeba may have a pathogenic role. At this point it is appropriate to wonder if microscopic examination of stool is still useful for the diagnosis of amoebiasis. The answer is yes, because it is fundamental to distinguish these three morphologically identical species from other amoebae that are certainly not pathogenic, so that patients do not receive unnecessary treatments that can both be harmful and lead to the development of drug resistance. Despite these problems regarding the identification of amoebae, microscopic identification of flagellates, ciliates and coccidia is relatively simple. As far intestinal microsporidia are concerned, given their small size (1-2.5 μm), correct identification can be achieved only by very experienced persons. An accurate parasitological examination requires not only an experienced microscopist but also:

-     High-quality reagents;

-     Standardization of analytical procedures, and their description in a clear and thorough manner in a manual available to all laboratory personnel;

-     Inclusion of positive and negative controls in each test session.

The aim of this work is to provide useful diagnostic support for the identification of protozoa, by describing the analytical procedures that can be easily carried out in the laboratory and by illustrating the microscopic findings through a rich iconography that shows parasites at different developmental stages, in both classic and atypical forms, as well as examples of "false parasites". The authors have decided to make this atlas freely usable online (, in both low and high resolution versions, permitting the printing of images at 300 dpi. The images and text of the atlas will be regularly updated. Anyone who intends to use this atlas is kindly asked to cite the source as follows: Giovanni Swierczynski*, Bruno Milanesi. "Atlas of human intestinal protozoa. Microscopic diagnosis".

* Corresponding author. E-Mail:


Every effort has been made to ensure that the advice and information in this atlas are true and accurate. However, neither the authors nor the persons who contributed in different ways to this work can be held legally responsible or liable for any errors or omissions in the text.