ABSTRACT
Rationale: It remains unclear how gastroesophageal reflux disease (GERD) affects allograft microbial community composition in lung transplant recipients and its impact on lung allograft inflammation and function. Objectives: Our objective was to compare the allograft microbiota in lung transplant recipients with or without clinically diagnosed GERD in the first year after transplant and assess associations between GERD, allograft microbiota, inflammation, and acute and chronic lung allograft dysfunction (ALAD and CLAD). Methods: A total of 268 BAL samples were collected from 75 lung transplant recipients at a single transplant center every 3 months after transplant for 1 year. Ten transplant recipients from a separate transplant center provided samples before and after antireflux Nissen fundoplication surgery. Microbial community composition and density were measured using 16S ribosomal RNA gene sequencing and quantitative polymerase chain reaction, respectively, and inflammatory markers and bile acids were quantified. Measurements and Main Results: We observed a range of allograft community composition with three discernible types (labeled community state types [CSTs] 1-3). Transplant recipients with GERD were more likely to have CST1, characterized by high bacterial density and relative abundance of the oropharyngeal colonizing genera Prevotella and Veillonella. GERD was associated with more frequent transitions to CST1. CST1 was associated with lower inflammatory cytokine concentrations than pathogen-dominated CST3 across the range of microbial densities observed. Cox proportional hazard models revealed associations between CST3 and the development of ALAD/CLAD. Nissen fundoplication decreased bacterial load and proinflammatory cytokines. Conclusions: GERD was associated with a high bacterial density, Prevotella- and Veillonella-dominated CST1. CST3, but not CST1 or GERD, was associated with inflammation and early development of ALAD and CLAD. Nissen fundoplication was associated with a reduction in microbial density in BAL fluid samples, especially the CST1-specific genus, Prevotella.
Subject(s)
Gastroesophageal Reflux , Lung Transplantation , Microbiota , Humans , Retrospective Studies , Gastroesophageal Reflux/complications , Lung , Inflammation , AllograftsABSTRACT
BACKGROUND: Oral and gut microbiomes have emerged as potential biomarkers in cancer. We characterised the oral and gut microbiomes in a prospective observational cohort of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients and evaluated the impact of chemoradiotherapy (CRT). METHODS: Saliva, oropharyngeal swabs over the tumour site and stool were collected at baseline and post-CRT. 16S RNA and shotgun metagenomic sequencing were used to generate taxonomic profiles, including relative abundance (RA), bacterial density, α-diversity and ß-diversity. RESULTS: A total of 132 samples from 22 patients were analysed. Baseline saliva and swabs had similar taxonomic composition (R2 = 0.006; p = 0.827). Oropharyngeal swabs and stool taxonomic composition varied significantly by stage, with increased oral RA of Fusobacterium nucleatum observed in stage III disease (p < 0.05). CRT significantly reduced the species richness and increased the RA of gut-associated taxa in oropharyngeal swabs (p < 0.05), while it had no effect in stool samples. These findings remained significant when adjusted by stage, smoking status and antibiotic use. CONCLUSIONS: Baseline oral and gut microbiomes differ by stage in this HPV+ cohort. CRT caused a shift towards a gut-like microbiome composition in oropharyngeal swabs. Stage-specific features and the transitions in oral microbiome might have prognostic and therapeutic implications.
Subject(s)
Chemoradiotherapy/adverse effects , Gastrointestinal Microbiome , Mouth Mucosa/microbiology , Oropharyngeal Neoplasms/therapy , Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , Saliva/microbiology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Carcinoma, Squamous Cell/virology , Female , Follow-Up Studies , Humans , Male , Mouth Mucosa/drug effects , Mouth Mucosa/radiation effects , Oropharyngeal Neoplasms/pathology , Oropharyngeal Neoplasms/virology , Papillomavirus Infections/virology , Prognosis , Prospective Studies , Saliva/drug effects , Saliva/radiation effectsABSTRACT
BACKGROUND/OBJECTIVES: Low-grade chronic inflammation in visceral adipose tissue and the intestines are important drivers of obesity associated insulin resistance. Bioactive compounds derived from plants are an important source of potential novel therapies for the treatment of chronic diseases. In search for new immune based treatments of obesity associated insulin resistance, we screened for tissue relevant anti-inflammatory properties in 20 plant-based extracts. METHODS: We screened 20 plant-based extracts to assess for preferential production of IL-10 compared to TNFα, specifically targetting metabolic tissues, including the visceral adipose tissue. We assessed the therapeutic potential of the strongest anti-inflammatory compound, indigo, in the C57BL/6J diet-induced obesity mouse model with supplementation for up to 16 weeks by measuring changes in body weight, glucose and insulin tolerance, and gut barrier function. We also utilized flow cytometry, quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and histology to measure changes to immune cells populations and cytokine profiles in the intestine, visceral adipose tissue (VAT), and liver. 16SrRNA sequencing was performed to examine gut microbial differences induced by indigo supplementation. RESULTS: We identifed indigo, an aryl hydrocarbon receptor (AhR) ligand agonist, as a potent inducer of IL-10 and IL-22, which protects against high-fat diet (HFD)-induced insulin resistance and fatty liver disease in the diet-induced obesity model. Therapeutic actions were mechanistically linked to decreased inflammatory immune cell tone in the intestine, VAT and liver. Specifically, indigo increased Lactobacillus bacteria and elicited IL-22 production in the gut, which improved intestinal barrier permeability and reduced endotoxemia. These changes were associated with increased IL-10 production by immune cells residing in liver and VAT. CONCLUSIONS: Indigo is a naturally occurring AhR ligand with anti-inflammatory properties that effectively protects against HFD-induced glucose dysregulation. Compounds derived from indigo or those with similar properties could represent novel therapies for diseases associated with obesity-related metabolic tissue inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Indigo Carmine/pharmacology , Insulin Resistance/physiology , Obesity/metabolism , Receptors, Aryl Hydrocarbon/agonists , Animals , Cytokines/metabolism , Diet, High-Fat , Gastrointestinal Microbiome , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Plant Extracts/chemistryABSTRACT
Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.
Subject(s)
High-Throughput Nucleotide Sequencing , Plant Diseases , Computational Biology , Double-Blind Method , Reproducibility of ResultsABSTRACT
BACKGROUND: Diarrhoea still accounts for considerable mortality and morbidity worldwide. The highest burden is concentrated in tropical areas where populations lack access to clean water, adequate sanitation and hygiene. In contrast to acute diarrhoea (<14 days), the spectrum of pathogens that may give rise to persistent diarrhoea (≥14 days) and persistent abdominal pain is poorly understood. It is conceivable that pathogens causing neglected tropical diseases play a major role, but few studies investigated this issue. Clinical management and diagnostic work-up of persistent digestive disorders in the tropics therefore remain inadequate. Hence, important aspects regarding the pathogenesis, epidemiology, clinical symptomatology and treatment options for patients presenting with persistent diarrhoea and persistent abdominal pain should be investigated in multi-centric clinical studies. METHODS/DESIGN: This multi-country, prospective, non-experimental case-control study will assess persistent diarrhoea (≥14 days; in individuals aged ≥1 year) and persistent abdominal pain (≥14 days; in children/adolescents aged 1-18 years) in up to 2000 symptomatic patients and 2000 matched controls. Subjects from Côte d'Ivoire, Indonesia, Mali and Nepal will be clinically examined and interviewed using a detailed case report form. Additionally, each participant will provide a stool sample that will be examined using a suite of diagnostic methods (i.e., microscopic techniques, rapid diagnostic tests, stool culture and polymerase chain reaction) for the presence of bacterial and parasitic pathogens. Treatment will be offered to all infected participants and the clinical treatment response will be recorded. Data obtained will be utilised to develop patient-centred clinical algorithms that will be validated in primary health care centres in the four study countries in subsequent studies. DISCUSSION: Our research will deepen the understanding of the importance of persistent diarrhoea and related digestive disorders in the tropics. A diversity of intestinal pathogens will be assessed for potential associations with persistent diarrhoea and persistent abdominal pain. Different diagnostic methods will be compared, clinical symptoms investigated and diagnosis-treatment algorithms developed for validation in selected primary health care centres. The findings from this study will improve differential diagnosis and evidence-based clinical management of digestive syndromes in the tropics. TRIAL REGISTRATION: ClinicalTrials.gov; identifier: NCT02105714 .
Subject(s)
Diarrhea/epidemiology , Abdominal Pain/etiology , Adolescent , Animals , Case-Control Studies , Child , Child, Preschool , Clinical Laboratory Techniques/economics , Clinical Laboratory Techniques/standards , Cost-Benefit Analysis , Cote d'Ivoire/epidemiology , Diarrhea/complications , Diarrhea/diagnosis , Diarrhea/economics , Diarrhea/microbiology , Diarrhea/parasitology , Feces/parasitology , Female , Humans , Indonesia/epidemiology , Infant , Infant, Newborn , Mali/epidemiology , Nepal/epidemiology , Prospective Studies , Risk FactorsABSTRACT
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.
Subject(s)
Closterovirus/isolation & purification , Genome, Viral/genetics , Plant Diseases/virology , Vitis/virology , Base Sequence , Closterovirus/genetics , Closterovirus/immunology , Closterovirus/ultrastructure , Enzyme-Linked Immunosorbent Assay , High-Throughput Nucleotide Sequencing , Molecular Sequence Data , Phylogeny , Plant Leaves/virology , RNA, Viral/chemistry , RNA, Viral/genetics , Sequence Analysis, RNAABSTRACT
The reuse of domestic and industrial wastewater in urban settings of the developing world may harm the health of people through direct contact or via contaminated urban agricultural products and drinking water. We assessed chemical and microbial pollutants in 23 sentinel sites along the wastewater and faecal sludge management and reuse chain of Kampala, Uganda. Water samples were examined for bacteria (thermotolerant coliforms (TTCs), Escherichia coli and Salmonella spp.) and helminth eggs. Physico-chemical parameters were determined. Water, sediment and soil samples and edible plants (yams and sugar cane) were tested for heavy metals. Water samples derived from the Nakivubo wetland showed mean concentrations of TTCs of 2.9 × 10(5) colony-forming units (CFU)/100 mL. Mean E. coli was 9.9 × 10(4) CFU/100 mL. Hookworm eggs were found in 13.5% of the water samples. Mean concentrations of iron (Fe), copper (Cu) and cadmium (Cd) were 21.5, 3.3 and 0.14 mg/L, respectively. In soil samples, we found a mean lead (Pb) concentration of 132.7 mg/L. In yams, concentrations of Cd, chromium (Cr) and Pb were 4.4, 4.0 and 0.2 mg/L, while the respective concentrations in sugar cane were 8.4, 4.3 and 0.2 mg/L. TTCs and E. coli in the water, Pb in soil, and Cd, Cr and Pb in the plants were above national thresholds. We conclude that there is considerable environmental pollution in the Nakivubo wetland and the Lake Victoria ecosystem in Kampala. Our findings have important public health implications, and we suggest that a system of sentinel surveillance is being implemented that, in turn, can guide adequate responses.
Subject(s)
Environmental Pollutants/analysis , Escherichia coli/isolation & purification , Helminths , Metals, Heavy/analysis , Salmonella/isolation & purification , Water Microbiology , Agriculture , Animals , Cadmium/analysis , Chromium/analysis , Copper/analysis , Dioscorea/chemistry , Environmental Monitoring , Geologic Sediments/analysis , Lakes , Ovum , Plants/chemistry , Saccharum/chemistry , Soil/chemistry , Soil Pollutants/analysis , Uganda , Wastewater , Water/chemistry , Water Pollutants, Chemical/analysis , WetlandsABSTRACT
BACKGROUND: Control efforts of soil-transmitted helminthiases rely primarily on large scale administration of anthelminthic drugs. The assessment of drug efficacies and understanding of drug behavior is pivotal to the evaluation of treatment successes, both in preventive chemo-therapy programs as well as in research of novel treatment options. The current WHO guidelines recommend an interval of 14-21 days between the treatment and follow-up, yet no in-depth analysis of egg excretion patterns of Trichuris trichiura after treatment has been conducted to date. METHODS: Within the framework of a multi-country trial to assess the efficacy and safety of albendazole-ivermectin combination therapy vs albendazole monotherapy against T. trichiura infections, we conducted a study collecting daily stool samples over the period of 28 days post-treatment in 87 participants in Pak Khan, Lao PDR. Egg counts were derived by duplicate Kato-Katz on-site for T. trichiura, hookworm and Ascaris lumbricoides and stool sample aliquots were subsequently analyzed by qPCR for the detection of T. trichiura infections. Sensitivity and specificity was calculated for each day separately using data derived by Kato-Katz to determine the optimal timepoint at which to assess drug efficacy. RESULTS: Egg excretion patterns varied across treatment arms. For T. trichiura, only the albendazole-ivermectin treatment led to a considerable reduction in mean egg counts, whereas both treatments reduced hookworm egg counts and A. lumbricoides were cleared in all participants after day 7. For T. trichiura, we found sensitivity to be highest at days 18 and 22 when using egg counts as outcome and days 19 and 24 when using qPCR. Specificity was high (>0.9) from day 14 onwards. For hookworm, the highest sensitivity and specificity were found at days 17 and 25, respectively. CONCLUSIONS: Based on our study, the ideal time period to assess drug efficacy for soil-transmitted helminth infections would be between day 18 and 24. The current WHO recommendation of 14 to 21 days is likely to yield acceptable outcome measures for soil-transmitted helminth infections. TRIAL REGISTRATION: NCT03527732.
Subject(s)
Anthelmintics , Helminthiasis , Trichuriasis , Animals , Humans , Albendazole/adverse effects , Ancylostomatoidea , Anthelmintics/therapeutic use , Feces , Helminthiasis/drug therapy , Ivermectin/therapeutic use , Soil , Trichuriasis/drug therapy , TrichurisABSTRACT
The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.
Subject(s)
Anopheles , Genetic Variation , Malaria, Falciparum , Plasmodium falciparum , Sporozoites , Plasmodium falciparum/genetics , Animals , Anopheles/parasitology , Sporozoites/genetics , Humans , Malaria, Falciparum/parasitology , Malaria, Falciparum/transmission , Malaria, Falciparum/blood , Mosquito Vectors/parasitology , Genotype , Host-Parasite Interactions/genetics , FemaleABSTRACT
The composition and function of the intestinal microbiota are major determinants of human health and are strongly influenced by diet, antibiotic treatment, lifestyle and geography. Nevertheless, we currently have only little data on microbiomes of non-westernized communities. We assess the stool microbiota composition in 59 children aged 2-5 years from the Adadle district of Ethiopia, Somali Regional State. Here, milk and starch-rich food are predominant components of the local diet, where the inhabitants live a remote, traditional agropastoral lifestyle. Microbiota composition, function and the resistome were characterized by both 16S rRNA gene amplicon and shotgun metagenomic sequencing and compared to 1471 publicly available datasets from children living in traditional, transitional, and industrial communities with different subsistence strategies. Samples from the Adadle district are low in Bacteroidaceae, and Prevotellaceae, the main bacterial representatives in the feces of children living in industrialized and non-industrialized communities, respectively. In contrast, they had a higher relative abundance in Streptococcaceae, Bifidobacteriaceae and Erysipelatoclostridiaceae. Further, genes involved in degradation pathways of lactose, D-galactose and simple carbohydrates were enriched. Overall, our study revealed a unique composition of the fecal microbiota of these agropastoral children, highlighting the need to further characterize the fecal bacterial composition of human populations living different lifestyles.
Subject(s)
Gastrointestinal Microbiome , Humans , Child , Gastrointestinal Microbiome/genetics , RNA, Ribosomal, 16S/genetics , Ethiopia , Bacteria/genetics , Feces/microbiology , Feeding BehaviorABSTRACT
Soil-transmitted helminth infections represent a large burden with over a quarter of the world's population at risk. Low cure rates are observed with standard of care (albendazole); therefore, a more effective combination therapy (albendazole and ivermectin) is being investigated but showed variable treatment efficacies without evidence of intrinsic parasite resistance. Here, we analyzed the microbiome of Trichuris trichiura and hookworm-infected patients and found an association of different enterotypes with treatment efficacy. 80 T. trichiura-infected patients with hookworm co-infections from Pak-Khan, Laos, received either albendazole (n = 41) or albendazole and ivermectin combination therapy (n = 39). Pre-/post-treatment stool samples were collected to monitor treatment efficacy and microbial communities were profiled using 16S rRNA gene sequencing, qPCR, and shotgun sequencing. We identified three bacterial enterotypes and show that pre-treatment enterotype is associated with efficacy of the combination treatment for both T. trichiura (CRET1 = 5.8%; CRET2 = 16.6%; CRET3 = 68.8%) and hookworm (CRET1 = 31.3%; CRET2 = 16.6%; CRET3 = 78.6%). This study shows that pre-treatment enterotype enables predicting treatment outcome of combination therapy for T. trichiura and hookworm infections.Trial registration: ClinicalTrials.gov, NCT03527732. Registered 17 May 2018, https://clinicaltrials.gov/ct2/show/NCT03527732 .
Subject(s)
Anthelmintics , Helminthiasis , Microbiota , Trichuriasis , Albendazole/therapeutic use , Anthelmintics/therapeutic use , Feces/parasitology , Helminthiasis/drug therapy , Humans , Ivermectin/therapeutic use , Parasite Egg Count , RNA, Ribosomal, 16S/genetics , Soil/parasitology , Trichuriasis/drug therapyABSTRACT
Short-read sequencing can provide detection of multiple genomic determinants of antimicrobial resistance from single bacterial genomes and metagenomic samples. Despite its increasing application in human, animal, and environmental microbiology, including human clinical trials, the performance of short-read Illumina sequencing for antimicrobial resistance gene (ARG) detection, including resistance-conferring single nucleotide polymorphisms (SNPs), has not been systematically characterized. Using paired-end 2 × 150 bp (base pair) Illumina sequencing and an assembly-based method for ARG prediction, we determined sensitivity, positive predictive value (PPV), and sequencing depths required for ARG detection in an Escherichia coli isolate of sequence type (ST) 38 spiked into a synthetic microbial community at varying abundances. Approximately 300,000 reads or 15× genome coverage was sufficient to detect ARGs in E. coli ST38, with comparable sensitivity and PPV to ~100× genome coverage. Using metagenome assembly of mixed microbial communities, ARG detection at E. coli relative abundances of 1% would require assembly of approximately 30 million reads to achieve 15× target coverage. The minimum sequencing depths were validated using public data sets of 948 E. coli genomes and 10 metagenomic rectal swab samples. A read-based approach using k-mer alignment (KMA) for ARG prediction did not substantially improve minimum sequencing depths for ARG detection compared to assembly of the E. coli ST38 genome or the combined metagenomic samples. Analysis of sequencing depths from recent studies assessing ARG content in metagenomic samples demonstrated that sequencing depths had a median estimated detection frequency of 84% (interquartile range: 30%-92%) for a relative abundance of 1%. IMPORTANCE Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15× coverage for ARG detection for E. coli in metagenomes. We demonstrate that for low relative abundances, sequencing depths of ~30 million reads or more may be required for adequate sensitivity for many applications.
Subject(s)
Anti-Bacterial Agents , Metagenome , Animals , Humans , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , High-Throughput Nucleotide Sequencing/methods , Metagenome/genetics , Genome, BacterialABSTRACT
BACKGROUND: Schistosomiasis 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 FINDINGS: We 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). CONCLUSION: A 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.
Subject(s)
Anthelmintics/therapeutic use , Drug Evaluation, Preclinical , Larva/growth & development , Schistosoma mansoni/drug effects , Schistosoma mansoni/growth & development , Animals , Female , Humans , Mice , Praziquantel/therapeutic use , Schistosomiasis mansoni/drug therapy , SerumABSTRACT
When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate high-resolution taxonomic and functional information at once. However, the technique is limited by missing information about host-to-microbe ratios observed in different body compartments. This limitation makes it difficult to plan shotgun sequencing assays, especially in the context of high sample multiplexing and limited sequencing output and is of particular importance for studies employing the recently described shallow shotgun sequencing technique. In this study, we evaluated the use of a quantitative PCR (qPCR)-based assay to predict host-to-microbe ratio prior to sequencing. Combining a two-target assay involving the bacterial 16S rRNA gene and the human beta-actin gene, we derived a model to predict human-to-microbe ratios from two sample types, including stool samples and oropharyngeal swabs. We then validated it on two independently collected sample types, including rectal swabs and vaginal secretion samples. This assay enabled accurate prediction in the validation set in a range of sample compositions between 4% and 98% nonhuman reads and observed proportions varied between -18.8% and +19.2% from the expected values. We hope that this easy-to-use assay will help researchers to plan their shotgun sequencing experiments in a more efficient way. IMPORTANCE When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate large amounts of data. However, in sample compositions with low or variable microbial density, shallowing sequencing can negatively affect microbial community metrics. Here, we show that variable sequencing depth decreases measured alpha diversity at differing rates based on community composition. We then derived a model that can determine sample composition prior to sequencing using quantitative PCR (qPCR) data and validated the model using a separate sample set. We have included a tool that uses this model to be available for researchers to use when gauging shallow sequencing viability of samples.
ABSTRACT
BACKGROUND: It is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data. RESULTS: Using three mock communities across a range of densities ranging from 8E+ 02 to 8E+ 09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+ 05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+ 04 and 8E+ 06 16S copies/ml. For the lowest densities, below 8E+ 03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples. CONCLUSION: Relative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+ 04 16S copies/ml.
Subject(s)
Bacteria/classification , Bronchoalveolar Lavage Fluid/microbiology , DNA Contamination , False Positive Reactions , Lung/microbiology , Microbiota/genetics , Humans , RNA, Ribosomal, 16S/geneticsABSTRACT
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.
Subject(s)
Bacteria/classification , Wastewater/microbiology , Whole Genome Sequencing/methods , Bacteria/genetics , Bacteria/isolation & purification , Environmental Monitoring , Genome, Bacterial , Metagenomics , Phylogeny , UgandaABSTRACT
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, 24â¯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 (24â¯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 24â¯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.
Subject(s)
Anthelmintics/adverse effects , Bacteria/drug effects , Gastrointestinal Microbiome/drug effects , Hookworm Infections/drug therapy , Adolescent , Albendazole/adverse effects , Albendazole/therapeutic use , Anthelmintics/administration & dosage , Anthelmintics/therapeutic use , Ascariasis/drug therapy , Ascariasis/epidemiology , Ascariasis/parasitology , Bacteria/genetics , Bacteria/isolation & purification , Bacteroidetes/genetics , Bacteroidetes/isolation & purification , Biotin/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Drug Therapy, Combination/adverse effects , Feces/microbiology , Gastrointestinal Microbiome/genetics , Hookworm Infections/epidemiology , Humans , Ivermectin/adverse effects , Ivermectin/therapeutic use , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Parasite Egg Count , Phenylenediamines/adverse effects , Phenylenediamines/therapeutic use , Pyrantel Pamoate/adverse effects , Pyrantel Pamoate/analogs & derivatives , Pyrantel Pamoate/therapeutic use , RNA, Ribosomal, 16S , Trichuriasis/drug therapy , Trichuriasis/epidemiology , Trichuriasis/parasitologyABSTRACT
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. TRIAL REGISTRATION: International Standard Randomised Controlled Trial, number ISRCTN15280205 .
Subject(s)
Anthelmintics/administration & dosage , Gastrointestinal Microbiome/genetics , Praziquantel/administration & dosage , Schistosoma mansoni/drug effects , Adolescent , Animals , Anthelmintics/therapeutic use , Biodiversity , Child , Child, Preschool , Cote d'Ivoire , DNA Barcoding, Taxonomic , Feces/parasitology , Female , Fusobacterium/drug effects , Fusobacterium/genetics , Fusobacterium/isolation & purification , Host-Parasite Interactions/drug effects , Humans , Male , Praziquantel/therapeutic use , RNA, Ribosomal, 16S/genetics , Schistosoma mansoni/genetics , Schistosomiasis mansoni/drug therapy , Schistosomiasis mansoni/epidemiology , Treatment OutcomeABSTRACT
BACKGROUND: Viruses belonging to the Flaviviridae and Bunyaviridae families show considerable genetic diversity. However, this diversity is not necessarily taken into account when developing diagnostic assays, which are often based on the pairwise alignment of a limited number of sequences. Our objective was to develop and evaluate a bioinformatics workflow addressing two recurrent issues of molecular assay design: (i) the high intraspecies genetic diversity in viruses and (ii) the potential for cross-reactivity with close relatives. METHODOLOGY: The workflow developed herein was based on two consecutive BLASTn steps; the first was utilized to select highly conserved regions among the viral taxon of interest, and the second was employed to assess the degree of similarity of these highly-conserved regions to close relatives. Subsequently, the workflow was tested on a set of eight viral species, including various strains from the Flaviviridae and Bunyaviridae families. PRINCIPAL FINDINGS: The genetic diversity ranges from as low as 0.45% variable sites over the complete genome of the Japanese encephalitis virus to more than 16% of variable sites on segment L of the Crimean-Congo hemorrhagic fever virus. Our proposed bioinformatics workflow allowed the selection-based on computing scores-of the best target for a diagnostic molecular assay for the eight viral species investigated. CONCLUSIONS/SIGNIFICANCE: Our bioinformatics workflow allowed rapid selection of highly conserved and specific genomic fragments among the investigated viruses, while considering up to several hundred complete genomic sequences. The pertinence of this workflow will increase in parallel to the number of sequences made publicly available. We hypothesize that our workflow might be utilized to select diagnostic molecular markers for higher organisms with more complex genomes, provided the sequences are made available.