Newly defined conditions for thein vitrocultivation and cryopreservation ofDientamoeba fragilis: new techniques set to fast track molecular studies on this organism

Abstract

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42°C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis.

Dientamoeba fragilis, the Neglected Trichomonad of the Human Bowel

“…Barratt et al (33) describe phenotypic differences between the in vitro growth characteristics of different clinical D. fragilis isolates and suggested that they might have a genetic basis. All D. fragilis isolates included in the study by Barratt et al (33) were confirmed to be genotype 1 isolates, so any potential genetic diversity must exist in other genes.…”

Enteric Protozoa in the Developed World: a Public Health Perspective

“…Microscopy of fixed smears with permanent staining (modified iron-hematoxylin or trichrome staining) (127,393,403) is considered the gold standard for diagnosis of D. fragilis infection, but this method is timeconsuming and relatively insensitive compared to molecular methods (391). More recently, diagnostic tests based on various conventional and real-time PCRs to detect the small-subunit (SSU) rRNA gene of D. fragilis have been developed to facilitate rapid, sensitive, and specific diagnosis in fresh stools (386,388,401,455), and the successful culture and cryopreservation of viable D. fragilis trophozoites from clinical specimens have been achieved (27,355). It was demonstrated that when microscopy is combined with other methods, the success of detection of D. fragilis is increased significantly (26,386,388).…”

Dientamoeba fragilis, One of the Neglected Intestinal Protozoa

“…The approach to in vitro culture is not new; however, some excellent improvements have recently been developed (39,40). Currently, no axenic cultures of D. fragilis exist.…”

“…A temperature of 42°C and a microaerophilic atmosphere are also optimum for growth. Compared to other media, Loeffler's slope medium led to much better growth of D. fragilis (40). A modified Earle's balanced salt solution containing cholesterol, ferric ammonium citrate, and rice starch is considered a superior liquid overlay that can be used along with Loeffler's serum slope for culture of D. fragilis under anaerobic conditions.…”