Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Abstract

Dientamoeba fragilis is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect D. fragilis DNA directly from human stool samples. The genetic diversity of D. fragilis from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that D. fragilis sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that D. fragilis shows remarkably little variation in its small-subunit rRNA gene.Dientamoeba fragilis is a protozoan parasite found in the mucosal crypts of the large intestines of humans. Originally D. fragilis was considered an ameba, but based on ultrastructural characteristics (2), antibody data (8), and phylogenetic data originating from 16S-like rRNA gene sequences, it has been established that it is a trichomonad (20), with no identified cyst stage. Most recent literature accepts that D. fragilis is an important enteric pathogen (7,10,18), with an estimated incidence of symptomatic infection of between 4 and 91% (11,21,25,26). Symptoms include abdominal pain, bloating, and diarrhea.Because of the lack of a cyst stage, diagnosis can be performed only on freshly passed stool or by the use of fixatives and permanent stains. In addition, day-to-day shedding is highly variable, which imposes the need for multiple sampling (24). These features have likely led to an underestimation of the prevalence of D. fragilis, which is reported to vary between 0.2% and more than 19% depending upon the population studied (3,9,15,16,26).Despite the relatively high prevalence of D. fragilis and its apparent role in patients presenting with gastrointestinal complaints, surprisingly little is known about its pathogenicity, route of transmission, epidemiology, and genetics. Because only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with nonpathogenic and pathogenic variants with similar morphology but different pathogenicities. This has been suggested for other lumendwelling protozoa such as Giardia duodenalis (13) and demonstrated for Entamoeba histolytica/Entamoeba dispar (4, 6). In a first report on variability in D. fragilis, the 16S-like ribosomal subunit DNA sequence of cultured D. fragilis from a small number of patients was analyzed by restriction fragment length polymorphism (RFLP) (14). Two of the 11 isolates gave a different restriction fragment pattern, indicating that there was genetic diversity in these D. fragilis cultures.The cumbersome and time-consuming techniques to maintain D. fragilis in culture in combination with the rapid disappearance of trophozoites from fresh feces have probably precluded more extensive and detailed studies of the genetic variability in D. fragilis. In addition, culture is not suitable for molecular ana...

Prospective Study of the Prevalence, Genotyping, and Clinical Relevance of Dientamoeba fragilis Infections in an Australian Population

“…The Australian isolates were found to be similar to those found in a recent study in the Netherlands and do not contain the polymorphic DdeI restriction site (CTTAG) at position 644 found in D. fragilis strain Bi/PA (15). RFLP analysis was undertaken on all 50 Australian samples to determine the genotypes present in the Australian population and the extent of genetic diversity.…”

“…to those found in a recent study in the Netherlands (15). The true incidence of the wild-type and variant forms in Australia needs to be established and to determine if such variation has any influence on the pathogenicity of the parasite.…”

“…Molecular genotyping and sequence analysis have demonstrated that D. fragilis exists as two genetically distinct forms (9,15,18,26). Stark et al (18) sequenced the SSU rRNA gene of seven Australian D. fragilis isolates, and the data generated from the seven showed no variation among them.…”

Dientamoeba fragilisis more prevalent thanGiardia duodenalisin children and adults attending a day care centre in Central Italy

“…The lack of an in vitro system to obtain highly purified nucleic acids from D. fragilis has limited the number of molecular studies on this parasite and prevented the use of a molecular approach to increase the detection level of this pathogen in faecal samples. To date, only PCR-derived methods based on the small-subunit rRNA gene has been developed (Peek et al, 2004;Johnson & Clark, 2000). The aim of this work was to evaluate the prevalence of infection with these pathogens and other associated parasites, in outpatients attending the day care centre of S. Silvestrini Hospital of Perugia (Central Italy) and to identify the genotypes of G. duodenalis by molecular analysis.…”

Cryptic diversity in intestinal protists

“…Subsequently, several groups have used PCR and either restriction fragment length polymorphism or sequencing to study the distribution and diversity of D. fragilis across the world. What is most remarkable is that with one exception, all infections studied since 2000 have involved the "new" genotype (Johnson & Clark, 2000;Peek et al, 2004;Windsor et al, 2004;Stark et al, 2005aStark et al, , 2005b. In other words, Bi/PA is not representative of the majority of D. fragilis circulating in the human population.…”