Dientamoeba fragilis : an emerging human pathogen

University of Technology, Sydney. Faculty of Science.Dientamoeba fragilis is a parasite of the human gastrointestinal tract that is often described as a cause of illness. Compared to other enteric protozoa of humans, information on D. fragilis is limited. Molecular studies on D. fragilis are particularly scarce and only three protein coding gene sequences are available for D. fragilis in Genbank. There is no published genome sequence for D. fragilis and knowledge on its metabolism, life cycle and mechanisms of pathogenicity is also lacking. Further research into these aspects of D. fragilis is warranted. Development of new diagnostic tests for D. fragilis has been a growing trend in recent years, though with a distinct focus on molecular diagnostics. Less attention has been paid to the development of antibody-based diagnostic tests for D. fragilis. While molecular assays are extremely sensitive and specific, antibody based assays are more applicable to routine diagnostic settings, mostly due to issues of cost and feasibility. Given the increasing importance of D. fragilis to human health, further research into the development of antibody-based diagnostic tests for D. fragilis is also warranted. This project was designed to address the shortcomings described previously. One major objective of this project was to sequence the transcriptome of D. fragilis (aim 2 of this project). This would dramatically increase the volume of molecular data available for D. fragilis and encourage future research on this organism. Another major objective was to develop polyclonal antibodies against a specific D. fragilis antigen which could be implemented in a diagnostic test (aim 3 of this project). To fulfil aims 2 and 3 of this project, long term cultures of D. fragilis would also be required to provide the necessary starting materials; D. fragilis RNA’s for sequencing and D. fragilis cells for the evaluation polyclonal antibodies. Therefore, the first objective was to improve current techniques for the cultivation of D. fragilis by evaluating various xenic culture systems for their ability to support D. fragilis growth (aim 1 of this project). Several media formulations were evaluated for their ability to support D. fragilis growth at various temperatures and atmospheric conditions. Different temperatures (from ambient room temperature to 42̊C) and various atmospheric conditions (aerobic, anaerobic and microaerophilic conditions) were also evaluated. Trophozoites were inoculated into fresh media under varying conditions and manual cell counts were performed daily to determine the media and conditions which supported optimal growth. Once optimal culture conditions were established, total RNA was extracted from culture sediments using TriSure reagent. As the culture systems employed were xenic (also containing a mixture of bacterial support flora), eukaryotic mRNA’s were enriched from total RNA extracts using oligo-(dT) cellulose chromatography. Purified eukaryotic mRNA’s were then subjected to next-generation sequencing. Upon assembly of the D. fragilis transcriptome, a suitable target protein was selected for polyclonal antibody development based on information obtained from the scientific literature. A homologue for this protein was identified in the D. fragilis transcriptome using blast searches. This nucleotide sequence then was translated into its amino acid sequence and immunogenic peptides were designed using this sequence as a template. Peptides were synthesised and conjugated to keyhole limpet hemocyanin for immunisation of rabbits. Anti-peptide antibodies were then purified from the resulting rabbit antisera by immunoaffinity chromatography. The resulting antibodies were evaluated by indirect fluorescent antibody testing (IFAT). Loeffler’s slope medium incubated at between 37̊C and 42̊C under microaerophilic conditions, supported significantly better growth of D. fragilis trophozoites compared to other media formulations and conditions. Next-generation sequencing of D. fragilis mRNA's generated 6,946 unique contigs. These contigs had an average length of 710 nucleotides with an average GC content of 34%. For the development of polyclonal antibodies, a protein from the enolase family was selected as a suitable target. Multiple enolase homologues were detected in the D. fragilis transcriptome and one of these was used as a template for the design of immunogenic peptides. Two of seven immunogenic peptides designed from this protein gave rise to two respective antibody preparations which reacted strongly to the surface of fixed D. fragilis trophozoites, as determined by the strong fluorescent signal observable in IFAT's. Each of the project aims was addressed succinctly and succesfully, to generate new knowledge on D. fragilis. Various culture systems were evaluated for their ability to support the growth of D. fragilis, and the optimal conditions (among those conditions tested) were determined. This knowledge will assist future investigators in the study of D. fragilis, as culture systems are an essential tool in the study of microorganisms. With the completion of the first D. fragilis transcriptome, thousands of unique nucleotide sequence can now be made available to future investigators. This work currently represents the largest scientific contribution to our understanding of D. fragilis molecular biology. Polyclonal antibodies against a surface antigen of D. fragilis were developed as a result of this project. These antibodies show great promise for implementation in an antibody-based diagnostic test for D. fragilis. Overall, this PhD project has contributed significantly to current knowledge on D. fragilis. It is hoped that this work will stimulate future research on D. fragilis so that our understanding of this organism continues to improve

The ambiguous life of Dientamoeba fragilis: the need to investigate current hypotheses on transmission.

Dientamoeba fragilis is an inhabitant of the human bowel and is associated with gastrointestinal illness. Despite its discovery over a century ago, the details of Dientamoeba's life cycle are unclear and its mode of transmission is unknown. Several theories exist which attempt to explain how Dientamoeba may be transmitted. One theory suggests that animals are responsible for the transmission of Dientamoeba. However, reports of Dientamoeba in animals are sporadic and most are not supported by molecular evidence. Another theory suggests that Dientamoeba may be transmitted via the ova of a helminth. Given that the closest relative of Dientamoeba is transmitted via the ova of a helminth, this theory seems plausible. It has also been suggested that Dientamoeba could be transmitted directly between humans. This theory also seems plausible given that other relatives of Dientamoeba are transmitted in this way. Despite numerous investigations, Dientamoeba's mode of transmission remains unknown. This review discusses the strengths and weaknesses of theories relating to Dientamoeba's mode of transmission and, by doing so, indicates where gaps in current knowledge exist. Where information is lacking, suggestions are made as to how future research could improve our knowledge on the life cycle of Dientamoeba

Detection of classic and cryptic Strongyloides genotypes by deep amplicon sequencing: A preliminary survey of dog and human specimens collected from remote Australian communities

© 2019 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Strongyloidiasis is caused by the human infective nematodes Strongyloides stercoralis, Strongyloides fuelleborni subsp. fuelleborni and Strongyloides fuelleborni subsp. kellyi. The zoonotic potential of S. stercoralis and the potential role of dogs in the maintenance of strongyloidiasis transmission has been a topic of interest and discussion for many years. In Australia, strongyloidiasis is prevalent in remote socioeconomically disadvantaged communities in the north of the continent. Being an isolated continent that has been separated from other regions for a long geological period, description of diversity of Australian Strongyloides genotypes adds to our understanding of the genetic diversity within the genus. Using PCR and amplicon sequencing (Illumina sequencing technology), we sequenced the Strongyloides SSU rDNA hyper-variable I and hyper-variable IV regions using Strongyloides-specific primers, and a fragment of the mtDNA cox1 gene using primers that are broadly specific for Strongyloides sp. and hookworms. These loci were amplified from DNA extracted from Australian human and dog faeces, and one human sputum sample. Using this approach, we confirm for the first time that potentially zoonotic S. stercoralis populations are present in Australia, suggesting that dogs represent a potential reservoir of human strongyloidiasis in remote Australian communities

A case-controlled study of Dientamoeba fragilis infections in children

Dientamoeba fragilis is a pathogenic protozoan parasite that is implicated as a cause of human diarrhoea. A case-controlled study was conducted to determine the clinical signs associated with D. fragilis infection in children presenting to a Sydney Hospital. Treatment options are also discussed. Stool specimens were collected from children aged 15 years or younger and analysed for the presence of D. fragilis. In total, 41 children were included in the study along with a control group. Laboratory diagnosis was performed by microscopy of permanently stained, fixed faecal smears and by real-time PCR. Gastrointestinal symptoms were present in 40/41 (98%) of these children with dientamoebiasis, with diarrhoea (71%) and abdominal pain (29%) the most common clinical signs. Chronic gastrointestinal symptoms were present in 2% of cases. The most common anti-microbial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. A treatment failure rate occurred in 15% of those treated with metronidazole. Follow-up treatment comprised of an additional course of metronidazole or iodoquinol was needed in order to achieve complete resolution of infection and symptoms in this group. This study demonstrates the pathogenic potential of D. fragilis in children and as such it is recommended that all laboratories must routinely test for this organism and treat if detected. © Cambridge University Press 2011

Activity of benzimidazoles against Dientamoeba fragilis (Trichomonadida, Monocercomonadidae) in vitro and correlation of beta-tubulin sequences as an indicator of resistance

Recently, Dientamoeba fragilis has emerged as a significant and common enteropathogen. The majority of patients with dientamoebiasis present with gastrointestinal complaints and chronic symptoms are common. Numerous studies have successfully demonstrated parasite clearance, coupled with complete resolution of clinical symptoms following treatment with various antiparasitic compounds. Despite this, there is very little in vitro susceptibility data available for the organism. Benzimidazoles are a class of antiparasitic drugs that are commonly used for the treatment of protozoan and helminthic infections. Susceptibility testing was undertaken on four D. fragilis clinical isolates against the following benzimidazoles: albendazole, flubendazole, mebendazole, nocodazole, triclabendazole and thiabendazole. The activities of the antiprotozoal compounds at concentrations ranging from 2 μg/mL to 500 μg/mL were determined via cell counts of D. fragilis grown in xenic culture. All tested drugs showed no efficacy. The beta-tubulin transcript was sequenced from two of the D. fragilis isolates and amino acid sequences predicted a susceptibility to benzimidazoles. This is the first study to report susceptibility profiles for benzimidazoles against D. fragilis, all of which were not active against the organism. This study also found that beta-tubulin sequences cannot be used as a reliable marker for resistance of benzimidazoles in D. fragilis. © D. Stark et al., published by EDP Sciences, 2014

Newly defined conditions for the in vitro cultivation and cryopreservation of Dientamoeba fragilis: New techniques set to fast track molecular studies on this organism

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 42C 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. © Cambridge University Press 2010

Machine learning and applications in microbiology

To understand the intricacies of microorganisms at the molecular level requires making sense of copious volumes of data such that it may now be humanly impossible to detect insightful data patterns without an artificial intelligence application called machine learning. Applying machine learning to address biological problems is expected to grow at an unprecedented rate, yet it is perceived by the uninitiated as a mysterious and daunting entity entrusted to the domain of mathematicians and computer scientists. The aim of this review is to identify key points required to start the journey of becoming an effective machine learning practitioner. These key points are further reinforced with an evaluation of how machine learning has been applied so far in a broad scope of real-life microbiology examples. This includes predicting drug targets or vaccine candidates, diagnosing microorganisms causing infectious diseases, classifying drug resistance against antimicrobial medicines, predicting disease outbreaks and exploring microbial interactions. Our hope is to inspire microbiologists and other related researchers to join the emerging machine learning revolution

A global genotyping survey of Strongyloides stercoralis and Strongyloides fuelleborni using deep amplicon sequencing

© 2019 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Strongyloidiasis is a neglected tropical disease caused by the human infective nematodes Strongyloides stercoralis, Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi. Previous large-scale studies exploring the genetic diversity of this important genus have focused on Southeast Asia, with a small number of isolates from the USA, Switzerland, Australia and several African countries having been genotyped. Consequently, little is known about the global distribution of geographic sub-variants of these nematodes and the genetic diversity that exists within the genus Strongyloides generally. We extracted DNA from human, dog and primate feces containing Strongyloides, collected from several countries representing all inhabited continents. Using a genotyping assay adapted for deep amplicon sequencing on the Illumina MiSeq platform, we sequenced the hyper-variable I and hyper-variable IV regions of the Strongyloides 18S rRNA gene and a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from these specimens. We report several novel findings including unique S. stercoralis and S. fuelleborni genotypes, and the first identifications of a previously unknown S. fuelleborni infecting humans within Australia. We expand on an existing Strongyloides genotyping scheme to accommodate S. fuelleborni and these novel genotypes. In doing so, we compare our data to all 18S and cox1 sequences of S. fuelleborni and S. stercoralis available in GenBank (to our knowledge), that overlap with the sequences generated using our approach. As this analysis represents more than 1,000 sequences collected from diverse hosts and locations, representing all inhabited continents, it allows a truly global understanding of the population genetic structure of the Strongyloides species infecting humans, non-human primates, and domestic dogs

Nonparametric Binary Classification to Distinguish Closely Related versus Unrelated P. Falciparum Parasites.

Assessing genetic relatedness of Plasmodium falciparum genotypes is a key component of antimalarial efficacy trials. Previous methods have focused on determining a priori definitions of the level of genetic similarity sufficient to classify two infections as sharing the same strain. However, factors such as mixed-strain infections, allelic suppression, imprecise typing methods, and heterozygosity complicate comparisons of apicomplexan genotypes. Here, we introduce a novel method for nonparametric statistical testing of relatedness for P. falciparum parasites. First, the background distribution of genetic distance between unrelated strains is computed. Second, a threshold genetic distance is computed from this empiric distribution of distances to demarcate genetic distances that are unlikely to have arisen by chance. Third, the genetic distance between paired samples is computed, and paired samples with genetic distances below the threshold are classified as related. The method is designed to work with any arbitrary genetic distance definition. We validated this procedure using two independent approaches to calculating genetic distance. We assessed the concordance of the novel nonparametric classification with a gold-standard Bayesian approach for 175 pairs of recurrent P. falciparum episodes from previously published malaria efficacy trials with microsatellite data from five studies in Guinea and Angola. The novel nonparametric approach was 98% sensitive and 84-89% specific in correctly identifying related genotypes compared with a gold-standard Bayesian algorithm. The approach provides a unified and systematic method to statistically assess relatedness of P. falciparum parasites using arbitrary genetic distance methodologies

Corrigendum: Angiostrongylus cantonensis: A review of its distribution, molecular biology and clinical significance as a human pathogen (Parasitology (2016) 143 (1087-1118) DOI: 10.1017/S0031182016000652)

© Cambridge University Press 2019. The authors apologise for an error in the manuscript: on page 1096, it was stated that most cases of angiostrongyliasis reported by Hochberg and colleagues (Hochberg et al., 2007, 2011) occurred in Honolulu, on the main island of Hawaii (Barratt et al., 2016). This is incorrect. The sentence should state that the highest incidence of cases reported by Hochberg and colleagues occurred on the island of Hawaii, which is the largest of the Hawaiian Islands. The Hawaii state capital of Honolulu is located on the island of Oahu, which is the most populous and third largest Hawaiian Island. We gratefully acknowledge Kirsten Snook (University of Hawaii at Hilo) and Dr Sarah Park (Hawaii Department of Health) who notified us independently of this error