**8. Diagnosis of dientamoebiasis**

While it is difficult to identify the trophozoites of *D. fragilis* morphologically, the only diag‐ nostic tool used to detect *D. fraglis* is microscopy using permanent stained smears. A large variety of stains have been used for the microscopic examination of *E. fragilis*. However, the most commonly used that also give much clearer images of the parasites are the trichrome and the iron-hematoxylin stains. The sample should be fixed immediately after staining to avoid degeneration of the trophozoites and staining should also occur sooner [107]. Trophozoites range in size from 5 to 15 μm in length, from 9 to 12 μm in width, normally with 1–2 fragmented nuclei with visible holes seen through the nucleus center. Smears may also contain tropho‐ zoites with the typical four nucleated form. No cyst stage has been recovered yet from humans despite being observed in mice [23]. Even under ideal conditions, with prompt preservation of stool and evaluation of permanent stained smears by experienced microscopists, Stark et al. (2010a and 2011) reported a sensitivity of 34% and 38%, respectively, compared to PCR (realtime and multiplex tandem—PCR) [10, 132]. Despite numerous studies reporting common occurrence of *D. fragilis* infection, no clinical antigen-based, molecular, or serologic diagnostics have been commercially developed to aid with laboratory identification to date, although current molecular based methods are used for research [133]. The culture of *D. fragilis* has been reported and is done in similar conditions as that of *E. histolytica*. Liquid or diphasic media is used that can be in xenic or axenic conditions [10]. Another diphasic medium based on the Loeffler's slope has also been demonstrated, and Earle's balanced salt solution (EBSS) has been successfully used for the growth of *D. fragilis* [23].

Molecular diagnostic methods have been very instrumental for the improvement of our understanding of different infections. There has been a significant gain in the development of molecular methods for the detection of *D. fragilis*, although compared to other organisms, this improvement has been much slower [32]. Several PCR protocols have been developed for the detection of this organism mainly for research laboratories. These protocols vary from conventional PCR to real-time PCR with increased sensitivity and specificity. Primers based on the small ribosomal RNA gene have been developed for this purpose [9]. Verweij and colleagues have developed a real-time PCR protocol using the 5.8S ribosomal RNA gene and they showed that this method was both specific and sensitive [28]. A variation of PCR based on the amplification of the internal transcribed spacer 1 region of *D. fragilis* has also been used for the molecular characterization of the parasite [130]. The actin gene has also been used as a target for the molecular characterization of this parasite [128]. Generally, the detection and/or the molecular characterization of the parasite begin with DNA purification, which is a very important and critical step in the amplification of the organism. Following DNA purification, the PCR master mixed is prepared depending on the procedure to be used. In the case of detection, the PCR protocol is generally sufficient. However, the molecular characterization often requires a sequencing step with or without the purification of the PCR amplicons. Other methods that have been used so far for the molecular characterization of *D. fragilis* include high-resolution melt curve analysis (HRM) and restriction fragment length polymorphism after amplification by PCR [9, 22].

Using HRM, Hussein and colleagues found 4 genetic profiles of which the first and most common profile and the last profile (Profile 4) were more associated with diarrhea compared to the two middle profiles [22]. However, the ITS showed two major genotypes although there were subgenotypes among those main categories. In another study, the ITS-1-5.8S rRNA gene-ITS-2 region of *D. fragilis* was found to be highly variable and pyrosequencing method identified 11 different alleles of the ITS-1 sequence showing the limitation of this gene in the molecular characterization of the parasite [130]. Briefly, the use of molecular methods has increased our knowledge on these organisms; much still remains to be discovered for the better understanding of issues related to pathogenicity, diagnosis, and prognosis.
