**3. Biology and life cycle of** *D. fragilis*

able for an active infection, concomitant with abdominal symptoms, nausea, and diarrhea. Moreover, controversy exists over the protective role of the parasite in priming the immune system in a beneficial way such as in selecting beneficial bacteria, keeping potential harmful microbial intruders at bay or producing metabolites beneficial to the host. Furthermore, the parasite's transmission mode remains a mystery. The microscopic identification and diagnoses of *D. fragilis*in stool requires skill and expertise; consequently, it is likely that many infections may go unidentified. Numerous studies have reported the effectiveness of treatment regimens using compounds such as paromomycin, hydroxyquinolines, and 5-nitroimidazoles, includ‐ ing metronidazole and tinidazole in the parasite eradication and the resolution of clinical symptoms. In addition, there is very little *in vitro* susceptibility data available for this parasite, making some current treatment options questionable. This chapter reviews the scientific literature relating to Dientamoeba's life cycle, prevalence, diagnosis, and pathogenicity.

*D. fragilis* is a ubiquitous protozoan parasite found in the gastrointestinal tract of humans. Electron microscopy [1] and molecular phylogenetic studies of the SSU rRNA gene [2,3] have recently confirmed the relationship of this parasite to trichomonads (lacking flagella). Al‐ though its pathogenic potential is still controversial, Jepps and Dobell in1918 were the first to report its pathogenicity when it was found to be the only agent detected in three patients with

**Figure 1.** Trophozoite of *D. fragilis* stained by iron hematoxylin stain (Photo by Adnan Al-Hindi, 2005). Photo extracted

Since then, many investigators have shown that patients infected with *D. fragilis* generally presented with bowel disorders with symptoms such as diarrhea, loose stools, and epigastric abdominal pains [6–12]. Furthermore, mounting evidence is accumulating reinforcing the pathogenicpotentialof*D.fragilis*[13–20].Lately,irritablebowel syndrome (IBS)hasbeenlinked to *D. fragilis* infections as a possible cause [21, 22], further underscoring its role in the causa‐ tion of disease. A great deal of controversy exists on the mode of transmission of *D. fragilis*, and while*Enterobius vermicularis*nematodehasbeenacceptedtoplaya role inits transmission,more

recently a report described the discovery of a new cyst stage in its life cycle [23].

**2. Recognition** *D. fragilis* **as a pathogen**

gastrointestinal clinical symptoms [5].

62 An Overview of Tropical Diseases

from Al-Hindi and Abu Shammala, 2013 [4].

Ranging in size from 5 to 15 μm in diameter, *D. fragilis* is a single-celled pleiomorphic troph‐ ozoite containing up to four nuclei [32, 20]. A large proportion of *D. fragilis* trophozoites are typically binucleated with a large, fragmented, central karyosome without peripheral chro‐ matin differentiated clearly in stained fecal smears [32,10]. Banik et al. (2012) have recently extensively described the surface structures and ultrasrtuctural details of *D. fragilis* popula‐ tions grown in xenic culture [31]. Using the scanning electron microscope, the group reported the existence of two different trophozoite populations—smooth and ruffled cells. Whether this represents a significant difference biologically or even in terms of the parasite's pathogenicity remains to be elucidated. Using the transmission electron microscope, neither mitochondria nor peroxisomes were reported [33, 34]. Nevertheless, a conspicuous organelle detected was the hydrogenosomes. Like many other organisms living in oxygen-deprived or anaerobic environments, these hydrogenosomes most probably represent the site of anaerobic respira‐ tion and energy production [35–39]. Different activities such as amoeboid movement, phago‐ cytosis, and bacterial adhesion to trophozoite surfaces were also reported by Banik and others (2012) [31]. Like many other parasitic protozoa such as *Trichomonas vaginalis*[40, 41, 33], *Giardia* [42], and *Leishmania* [43], virus-like particles (VLPs) have also been reported to be seen in *D. fragilis* trophozoites. Many groups have reported an association between the presence of VLPs within *T. vaginalis* and variations in protozoa phenotypes, virulence factors, and disease pathogenesis [44–46]. More details of the ultrastructure of *D. fragilis* are available in a review authored by Banik et al. (2012) [31].

The complete life cycle and the mode of transmission of *D. fragilis* remain ambiguous and equivocal. The only known stage thus far is the trophozoite (Fig. 2). Dobell (1940) was the first to predict *E. vermicularis* egg to act as a vector for the transmission of *D. fragilis* [47]. Recently, Roser et al. (2013) have detected *D. fragilis* DNA inside *E. vermicularis* eggs agreeing with the prediction of Dobell in 1940 [48]. While many reports of a higher than anticipated rate of coinfection between *D. fragilis* and E. vermicularis led researchers to postulate *E. vermicularis* as the probable vector responsible for its transmission [48, 49], other groups have proved no coinfections with *D. fragilis* and other worms, suggesting fecal-oral transmission as the possible mechanism of transmission of *D. fragilis*[9, 10]. A new study by Munasinghe et al. (2013) using

**Figure 2.** Life cycle of *D. fragilis*. Reproduced from: Centers for Disease Control and Prevention. DPDx: *Dientamoeba fragilis* infection. Available at: http://www.cdc.gov/dpdx/dientamoeba/index.html.

rodents and mice infected with human isolates reported the discovery of a new cyst stage in the life cycle of *D. fragilis* strongly suggesting oral–fecal transmission as the possible route of infection [23]. Moreover, Stark et al. (2014) have recently reported a cyst form of *D. fragilis*from human clinical samples, further supporting that cysts are likely to be the transmission forms [50]. The role of animals and zoonotic transmission of the parasite is still ambiguous despite a recent study reporting pigs and sheep as natural hosts of dientamoebiasis [51]. The reader is invited to read an excellent review on the topic written by Clark et al. (2014) [52].

## **4. Epidemiology of dientamoebiasis and its occurrence**

Since its hypothetical association with IBS and other bowel disorders, probable pathogenicity, and the existence of gaps in its life cycle and mode of transmission, many investigators have become increasingly aware of the importance of *D. fragilis*. This has led to the development of more sensitive diagnostic techniques for its proper identification and determination of its accurate prevalence. It is now recognized as being more prevalent than *Giardia* [7, 11, 25, 27, 29, 53–65]. Table 1 shows the prevalence rates of *D. fragilis* ranging between 0.3% and 90% in many countries worldwide. With the exception of few studies, light microscopy was the tool used in those studies. The use of more sensitive techniques such as PCR or cultivation may result in different and more accurate prevalence rates [10, 28, 66]. Unlike many parasitic infections, *D. fragilis* has been shown to have high infection rates in developed countries than in underprivileged countries [25, 67].


rodents and mice infected with human isolates reported the discovery of a new cyst stage in the life cycle of *D. fragilis* strongly suggesting oral–fecal transmission as the possible route of infection [23]. Moreover, Stark et al. (2014) have recently reported a cyst form of *D. fragilis*from human clinical samples, further supporting that cysts are likely to be the transmission forms [50]. The role of animals and zoonotic transmission of the parasite is still ambiguous despite a recent study reporting pigs and sheep as natural hosts of dientamoebiasis [51]. The reader is

**Figure 2.** Life cycle of *D. fragilis*. Reproduced from: Centers for Disease Control and Prevention. DPDx: *Dientamoeba*

Since its hypothetical association with IBS and other bowel disorders, probable pathogenicity, and the existence of gaps in its life cycle and mode of transmission, many investigators have become increasingly aware of the importance of *D. fragilis*. This has led to the development of more sensitive diagnostic techniques for its proper identification and determination of its accurate prevalence. It is now recognized as being more prevalent than *Giardia* [7, 11, 25, 27, 29, 53–65]. Table 1 shows the prevalence rates of *D. fragilis* ranging between 0.3% and 90% in many countries worldwide. With the exception of few studies, light microscopy was the tool used in those studies. The use of more sensitive techniques such as PCR or cultivation may result in different and more accurate prevalence rates [10, 28, 66]. Unlike many parasitic

invited to read an excellent review on the topic written by Clark et al. (2014) [52].

**4. Epidemiology of dientamoebiasis and its occurrence**

*fragilis* infection. Available at: http://www.cdc.gov/dpdx/dientamoeba/index.html.

64 An Overview of Tropical Diseases



Table adapted from Barratt et al. 2011. With kind permission from Dr. Damien Stark.

a MSM denotes men who have sex with men.

**Prevalence Sample source and type Number of**

5.5% Fecal specimens submitted to a university hospital in Tunisia

66 An Overview of Tropical Diseases

0.8% HIV negative MSMa

11.7% Patients suspected of infection with gut parasites; feces

14.6%; 16.9% Individuals attending complimentary health

2.1% HIV negative patients; feces 48 San Pedro Sula,

5.1% Routine testing; feces 857 Oman [59]

3% HIV-positive patients; feces 34 North Brazil [87] 11.3% Gastrointestinal tract patients; feces 151 Italy [61]

0.9% Diarrhea patients; feces 6750 Sydney, Australia [9] 0.82% Sanitary employees; feces 241 Malatya, Turkey [88] 6.3% Patients infected with a gut parasite; feces 448 Brussels, Belgium [6] 3.7% Gastrointestinal tract patients; feces 3139 Italy [58] 3.4% Gastrointestinal tract patients; feces 1141 Italy [57] 4.1% Gastrointestinal tract patients; feces 1989 Italy [53] 2% Children and neonates patients; feces 350 Surt, Libya [89] 2.7% Aborigines; feces 112 Salta, Argentina [90] 2.7% Feces 770 Turkey [91] 8.9% Patients infected with gut parasites; feces 168 Egypt [24] 29.8% Patients infected with gut parasites; feces 168 Egypt [24]

8.8% Admitted patients; feces 400 Turkey, Celal Bayar

0.3% HIV-positive MSM; feces 618 Sydney, Australia 1.1% Non-MSM patients; feces 622 Sydney, Australia

32% Bowel complaints patients; feces 397 Zwolle, The

0.2% School children; feces 2975 Van Province, Turkey [94] 5.2% Bowel complaints; feces 750 Sydney, Australia [11] 1.6% Digestive disorder patients; feces 8313 Catalonia, Spain [95]

care practices (2002–2004 and 2005–2007); feces

**patients**

; feces 628 Sydney, Australia [92]

**Country/region Reference**

[85]

[29]

[93]

Honduras

27053 Sfax, Tunisia [86]

University

103 Denmark [25]

Netherlands

3719; 2491 British Isles [67]

b In this Tabriz, Iran, study (Sarafraz et al., 2013), 26 samples were reported as suspicious cases in trichrome-stained smears.

**Table 1.** Global prevalence of *D. fragilis* infections in stool samples from various sources

Conflicting reports exist regarding the age-group distribution of *D. fragilis* infections. Two studies, Danish and Canadian, reported a high infection rate in subjects aged between 16 and 20 years,respectively [25, 60].On the other hand, despite being statistically insignificant,Rayan et al. (2007) reported a higher infection rate in individuals aged between 30 and 40 years [24]. In contrast, other investigators reported a higher incidence rate in children and in less than 20 years old [8, 16, 27, 29, 63, 74, 95, 100]. In a recent study by Al-Hindi and Abu Shammala (2013) in the Gaza strip regarding age, children less than 5 years of age were reported to have a prevalenceof11.3%,whiletheage-group20–26yearshad15.4%[4].This is incontrasttofindings byGirginkardesleretal.(2003),whoreportedthat*D.fragilis*infectionwashigheramongchildren than adult [29]. No plausible explanation to these variations in age distribution of *D. fragilis* incidences is proposed. Nevertheless, hygiene and modest sanitation have been suggested as likely to prejudice groups to infections with *D. fragilis* and other intestinal protozoa irrespec‐ tive of age making the fecal oral route as the probable route of transmission [12, 24, 53, 101]. With respect to the association between gender and *D. fragilis* infections, numerous studies reportdissimilartrends.While severalinvestigators reportmore infectionincidences infemales than males [16, 24, 55, 57, 86, 95], other studies describe a drift towards males in certain agegroups [4, 8, 60]. For more details on the subject, the reader is advised to consult the review by Barratt et al. (2011) [12].

### **5. Pathogenicity and clinical symptoms of dientamoebiasis**

Originally proposed as a pathogen in 1936 by Hakansson, there still remains some reluctance by many investigators accepting *D. fragilis* as a pathogen [102, 103]. For example, in a recent retrospective case-control study in the Netherlands elucidating the clinical importance of *D. fragilis* in children with chronic abdominal pain, De Jong et al. (2014) detected *D. fragilis* in 43.2% of patients with chronic abdominal pain and in 50.6% in the controls (without gastro‐ intestinal symptoms) (*p* = 0.255) [104]. Thus, there are no significant differences in symptoms comparing children with and without *D. fragilis* infection. Furthermore, no relation was found between clinical and microbiological response after treatment for *D. fragilis* in the same study, suggesting that there is no association between chronic abdominal pain *D. fragilis* infection. Nevertheless, many current studies have acknowledged and confirmed the pathogenic potential of *D. fragilis*. It is often detected in the feces of patients suffering from gastrointestinal tract disorders and presenting symptoms such as loose stools, diarrhea, urgency to defecate, vomiting, nausea, anorexia, weight loss, abdominal pain, and fever [6–9, 11, 21, 29, 105, 106]. Many investigators have reported the tendency for this parasite to cause persistent diarrhea [9, 55]. An example of a study confirming the pathogenic role of *D. fragilis* is an Italian study in 2007, where Crotti and D'Annibale found that between 3.4% and 4.1% of patients with various bowel complaints carried Dientamoeba [55, 57]. Another report corroborating the pathogenic potential of the organism is an Australian study in which 5.4% (35/650) of patients with bowel disorders were reported to have Dientamoeba in their stools, with 83.3% of them suffering from diarrhea [10]. Furthermore, Dientamoeba has been linked it with irritable bowel syndrome (IBS) [22, 97, 105]. Patients carrying Dientamoeba may also experience eosinophilia [10, 63, 64, 103, 106, 107].

### **6. Treatment of** *D. fragilis* **infections**

While still not recognized as a pathogen, the ability to resolve associated symptoms by eradicating *D. fragilis* using different drugs provides some proof for its possible pathogenic nature [6, 16, 20, 69, 102, 103, 108–110]. There is still no agreement as to the best regimen for the complete elimination of the organism. Stark et al. (2010b) and Preiss et al. (1990) reported a treatment ineffectiveness and/or relapse of dientamoebiasis following the use of metronida‐ zole only [11, 64]. In a recent Danish randomized trial, 96 children in Denmark with *D. fragilis* infection and chronic gastrointestinal symptoms were treated with a 10-day course of metronidazole or placebo [111]. Change in gastrointestinal symptoms following treatment did not differ significantly between the groups. Eradication of *D. fragilis* was significantly greater in the metronidazole group as assessed by PCR 2 weeks after completion of therapy, although PCR positivity rebounded by 8 weeks after completion of therapy to levels comparable with those seen in placebo recipients. The eradication of *D. fragilis* was significantly greater in the metronidazole group, although it declined rapidly from 62.5% 2 weeks after end of treatment to 24.9% 8 weeks after end of treatment. The findings of the study did not provide evidence to support routine metronidazole treatment of *D. fragilis*-positive children with chronic gastro‐ intestinal symptoms. However, the complete resolution of symptoms and elimination of the organism were noted following the administration of either iodoquinol, paromomycin, or a combination of the two [11, 107]. Most recently, Halkjær et al. (2015) described a case history of a 16-year-old Danish patient who had suffered severe abdominal discomfort and flatulence through his lifetime following infection with *D. fragilis*. The patient was treated initially with a high dose of metronidazole, which eradicated the parasite and kept him without symptoms for 1 year [112]. However, recurrence of the symptoms and recurrence of the *D. fragilis*infection were thereafter treated successfully with paromomycin [112]. Other drugs that are also reported to effectively eradicate the parasite leading to clinical cure included oxytetracycline, doxycycline, tinidazole, secnidazole, ornidazole, and erythromycin [29, 30, 64, 102, 113]. Despite the lack of randomized controlled trial data, the literature suggests paromomycin is a more efficacious agent than metronidazole [6, 11, 114]. New potential therapeutic compounds are constantly being screened for by investigators. More recently, Stark et al. (2014) have shown that there is no therapeutic response against dientamoebiasis with benzimidazoles (such as albendazole and mebendazole) [115].

groups [4, 8, 60]. For more details on the subject, the reader is advised to consult the review by

Originally proposed as a pathogen in 1936 by Hakansson, there still remains some reluctance by many investigators accepting *D. fragilis* as a pathogen [102, 103]. For example, in a recent retrospective case-control study in the Netherlands elucidating the clinical importance of *D. fragilis* in children with chronic abdominal pain, De Jong et al. (2014) detected *D. fragilis* in 43.2% of patients with chronic abdominal pain and in 50.6% in the controls (without gastro‐ intestinal symptoms) (*p* = 0.255) [104]. Thus, there are no significant differences in symptoms comparing children with and without *D. fragilis* infection. Furthermore, no relation was found between clinical and microbiological response after treatment for *D. fragilis* in the same study, suggesting that there is no association between chronic abdominal pain *D. fragilis* infection. Nevertheless, many current studies have acknowledged and confirmed the pathogenic potential of *D. fragilis*. It is often detected in the feces of patients suffering from gastrointestinal tract disorders and presenting symptoms such as loose stools, diarrhea, urgency to defecate, vomiting, nausea, anorexia, weight loss, abdominal pain, and fever [6–9, 11, 21, 29, 105, 106]. Many investigators have reported the tendency for this parasite to cause persistent diarrhea [9, 55]. An example of a study confirming the pathogenic role of *D. fragilis* is an Italian study in 2007, where Crotti and D'Annibale found that between 3.4% and 4.1% of patients with various bowel complaints carried Dientamoeba [55, 57]. Another report corroborating the pathogenic potential of the organism is an Australian study in which 5.4% (35/650) of patients with bowel disorders were reported to have Dientamoeba in their stools, with 83.3% of them suffering from diarrhea [10]. Furthermore, Dientamoeba has been linked it with irritable bowel syndrome (IBS) [22, 97, 105]. Patients carrying Dientamoeba may also experience eosinophilia

While still not recognized as a pathogen, the ability to resolve associated symptoms by eradicating *D. fragilis* using different drugs provides some proof for its possible pathogenic nature [6, 16, 20, 69, 102, 103, 108–110]. There is still no agreement as to the best regimen for the complete elimination of the organism. Stark et al. (2010b) and Preiss et al. (1990) reported a treatment ineffectiveness and/or relapse of dientamoebiasis following the use of metronida‐ zole only [11, 64]. In a recent Danish randomized trial, 96 children in Denmark with *D. fragilis* infection and chronic gastrointestinal symptoms were treated with a 10-day course of metronidazole or placebo [111]. Change in gastrointestinal symptoms following treatment did not differ significantly between the groups. Eradication of *D. fragilis* was significantly greater in the metronidazole group as assessed by PCR 2 weeks after completion of therapy, although PCR positivity rebounded by 8 weeks after completion of therapy to levels comparable with

**5. Pathogenicity and clinical symptoms of dientamoebiasis**

Barratt et al. (2011) [12].

68 An Overview of Tropical Diseases

[10, 63, 64, 103, 106, 107].

**6. Treatment of** *D. fragilis* **infections**
