Development and application of -omics and bioinformatics approaches for a deeper understanding of infectious diseases systems

Background: Research in infectious diseases underwent a revolution with the uprising of Omics approaches, including, but not limited to, genomics, metagenomics and metatranscriptomics. In fact, there are several examples where Omics approaches showed their potential to tackle different challenges related to the versatile nature of infectious diseases by promoting “studies of one” to “system-wide studies”. In the frame of this PhD programme, we focused on the development and validation of Omics approaches and bioinformatics workflow aiming at tackling mainly diagnostics but also to some extents the treatment of infectious diseases. The four applications presented in this thesis had following specific objectives; (i) to develop and validate a bioinformatics approach aiming at selecting high quality markers among a large amount of complete genomic sequences; (ii) to characterise the viral metagenome of a plant to determine aetiology of a disease that could not be identified and/or fully characterised with other tools; (iii) to assess the potential of metagenomics in the field of personalised medicine and compare its diagnostics accuracy with validated diagnostics tools; and (iv) to make a system-wide survey of microbial populations and estimate its potential to cause harm to humans. Methods: Methodology was specific for each application but as a general rule, we only used published bioinformatics tools that have been used and validated in other studies. This includes, but is not limited to, the BLAST algorithm for the comparison of sequences to various databases and the MIRA assembler to assemble the metagenomics datasets obtained within the different projects. Results: For clarity, the results are summarised by project, corresponding to the different applications investigated during this PhD. Project (i): The developed bioinformatics workflow allowed the selection of highly conserved and specific molecular markers among various viral species with inputs of up to several hundred complete genomic sequences. The quality of the selected markers was successfully validated using several types of molecular assays including real-time PCR, LAMP and Sanger sequencing. Project (ii): We were able to find the aetiology of a grapevine plant presenting leafroll symptoms. A new virus, named Grapevine Leafroll-associated virus 4 Ob, with a thirteen kilobases genome was found in the viral metagenome. Other viruses that were co-identified in the virome were known to be asymptomatic viruses for grapevine, and with the help of additional serological experiences, we were able to confirm that this GLRaV-4 Ob was the causative agent of the Leafroll symptoms. Project (iii): The gut pathobiomes from four patients presenting persistent digestive disorders were fully characterised using a metagenomics approach. Comparison of validated diagnostics tools with this approach showed that the diagnostics rate was in favour of the latter for the detection of bacterial and helminths pathogens and in favour of the validated tools for the detection of viruses and protozoa. Using the same datasets, but compared to a different database, we were also able to screen the stool samples for antimicrobial resistance genes and retrieve potential resistance genes that might interfere with the treatment of these patients. Project (iv): In this project, a system-wide assessment of the microbial communities of the wastewater treatment system was done using a metagenomics approach. We were able to demonstrate how closely the genetic diversity of Escherichia coli and the overall genetic diversity were linked in this environment. We were also able to map the repartition of different pathogenic classes, including bacteria, helminths, intestinal protozoa and viruses as well as to show if and how human waterborne pathogens spread throughout this ecosystem. Conclusion: Omics offer new strategies of how challenges, mainly related to the vast diversity within the research area of infectious diseases, can be tackled. Meta-analyses, like metagenomics or metatranscriptomics are the applications that benefited most from the use of Next-Generation Sequencing technologies, and they now allow system-wide studies where previous studies were only focusing on one parameter (one microbe or one specific gene for instance). However, these Omics approaches have their limitations, mainly due to the bioinformatics challenges they give rise to. As a general conclusion, it is foreseeable that, because of the increased amount of results they generate, Omics approaches, once matured, will be more widely used and will replace standard approaches in the field of infectious diseases

Investigations on the interplays between Schistosoma mansoni, praziquantel and the gut microbiome

Schistosomiasis is a neglected tropical disease burdening millions of people. One drug, praziquantel, is currently used for treatment and control. Clinically relevant drug resistance has not yet been described, but there is considerable heterogeneity in treatment outcomes, ranging from cure to only moderate egg reduction rates. The objectives of this study are to investigate potential worm-induced dysbacteriosis of the gut microbiota and to assess whether a specific microbiome profile could influence praziquantel response.; Using V3 and V4 regions of 16S rRNA genes, we screened the gut microbiota of 34 Schistosoma mansoni infected and uninfected children from Côte d'Ivoire. From each infected child one pre-treatment, one 24-hour and one 21-day follow-up sample after administering 60 mg/kg praziquantel or placebo, were collected.; Overall taxonomic profiling and diversity indicators were found to be close to a "healthy" gut structure in all children. Slight overall compositional changes were observed between S. mansoni-infected and non-infected children. Praziquantel treatment was not linked to a major shift in the gut taxonomic profiles, thus reinforcing the good safety profile of the drug by ruling out off-targets effects on the gut microbes.16S rRNA gene of the Fusobacteriales order was significantly more abundant in cured individuals, both at baseline and 24 hours post-treatment. A real-time qPCR confirmed the over-abundance of Fusobacterium spp. in cured children. Fusobacterium spp. abundance could also be correlated with treatment induced S. mansoni egg-reduction.; Our study suggests that neither a S. mansoni infection nor praziquantel administration triggers a significant effect on the microbial composition and that a higher abundance of Fusobacterium spp., before treatment, is associated with higher efficacy of praziquantel in the treatment of S. mansoni infections.; International Standard Randomised Controlled Trial, number ISRCTN15280205

Off-target effects of tribendimidine, tribendimidine plus ivermectin, tribendimidine plus oxantel-pamoate, and albendazole plus oxantel-pamoate on the human gut microbiota

Soil-transmitted helminths infect 1.5 billion people worldwide. Treatment with anthelminthics is the key intervention but interactions between anthelminthic agents and the gut microbiota have not yet been studied. In this study, the effects of four anthelminthic drugs and combinations (tribendimidine, tribendimidine plus ivermectin, tribendimidine plus oxantel-pamoate, and albendazole plus oxantel-pamoate) on the gut microbiota were assessed. From each hookworm infected adolescent, one stool sample was collected prior to treatment, 24 h post-treatment and 3 weeks post-treatment, and a total of 144 stool samples were analyzed. The gut bacterial composition was analyzed using 16S rRNA gene sequencing. Tribendimidine given alone or together with oxantel-pamoate, and the combination of albendazole and oxantel pamoate were not associated with any major changes in the taxonomic composition of the gut microbiota in this population, at both the short-term post-treatment (24 h) and long-term post-treatment (3 weeks) periods. A high abundance of the bacterial phylum Bacteroidetes was observed following administration of tribendimidine plus ivermectin 24 h after treatment, due predominantly to difference in abundance of the families Prevotellaceae and Candidatus homeothermaceae. This effect is transient and disappears three weeks after treatment. Higher abundance of Bacteroidetes predicts an increase in metabolic pathways involved in the synthesis of B vitamins. This study highlights a strong relationship between tribendimidine and ivermectin administration and the gut microbiota and additional studies assessing the functional aspects as well as potential health-associated outcomes of these interactions are required

Qualitative microbiome profiling along a wastewater system in Kampala, Uganda

Kampala, the capital city of Uganda, is rapidly expanding without adequate wastewater treatment facilities to accommodate the current estimated population of 1.68 million people. Hence, freshwater bodies and natural ecosystems around the city are heavily polluted with organic and inorganic contaminants. Yet, there is a paucity of data on pathogenic microorganisms, which potentially threatens health of local communities. We performed a qualitative microbial analysis using a whole metagenome sequencing approach encompassing over 150 gigabases of sequencing data to characterize the Nakivubo wastewater system, which includes a wastewater channel and surrounding wetlands. We found that microbial diversity is heterogeneous throughout the system and that three community state types could be differentiated. We showed the presence of various waterborne agents of gastrointestinal infections in humans, which were associated with leakage occurring around two locations along the wastewater channel. Our data indicate that the microbial decontamination capacity of the local wastewater treatment facility was insufficient at the time of sampling, and that several areas of the wetlands were contaminated with human pathogens, indicating that parts of the wetlands are potentially unsafe for urban agriculture

Experiences and Lessons from a Multicountry NIDIAG Study on Persistent Digestive Disorders in the Tropics.

<p>Experiences and Lessons from a Multicountry NIDIAG Study on Persistent Digestive Disorders in the Tropics</p

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Validation of a human-serum-based in vitro growth method for drug screening on juvenile development stages of Schistosoma mansoni.

BackgroundSchistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.Methodology/principal findingsWe compared inter-batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC50: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC50: 4.43 μM in vitro, 0.48 μM in vivo).ConclusionA large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts

Biological, serological, and molecular characterization of a hghly divergent strain of grapevine leafroll-associated virus 4 causing grapevine leafroll disease

The complete genome sequence of a highly divergent strain of Grapevine leafroll-associated virus 4 (GLRaV-4) was determined using 454 pyrosequencing technology. This virus, designated GLRaV-4 Ob, was detected in Vitis vinifera 'Otcha bala' from our grapevine virus collection at Agroscope. The GLRaV-4 Ob genome length and organization share similarities with members of subgroup II in the genus Ampelovirus (family Closteroviridae). Otcha bala was graft-inoculated onto indicator plants of cultivar Gamay to evaluate the biological properties of this new strain, and typical leafroll symptoms were induced. A monoclonal antibody for the rapid detection of GLRaV-4 Ob by enzyme-linked immunosorbent assay is available, thus facilitating large-scale diagnostics of this virus. Based on the relatively small size of the coat protein, the reduced amino acid identity and the distinct serological properties, our study clearly shows that GLRaV-4 Ob is a divergent strain of GLRaV-4. Furthermore, molecular and serological data revealed that the AA42 accession from which GLRaV-7 was originally reported is in fact co-infected with GLRaV-4 Ob and GLRaV-7. This finding challenges the idea that GLRaV-7 is a leafroll-causing agent