Organoids and bioengineered intestinal models: Potential solutions to the Cryptosporidium culturing dilemma

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes

Identification of Zoonotic Genotypes of Giardia duodenalis

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases

Molecular characterisation of protist parasites in human-habituated mountain gorillas (Gorilla beringei beringei), humans and livestock, from Bwindi impenetrable National Park, Uganda

Over 60 % of human emerging infectious diseases are zoonotic, and there is growing evidence of the zooanthroponotic transmission of diseases from humans to livestock and wildlife species, with major implications for public health, economics, and conservation. Zooanthroponoses are of relevance to critically endangered species; amongst these is the mountain gorilla (Gorilla beringei beringei) of Uganda. Here, we assess the occurrence of Cryptosporidium, Cyclospora, Giardia, and Entamoeba infecting mountain gorillas in the Bwindi Impenetrable National Park (BINP), Uganda, using molecular methods. We also assess the occurrence of these parasites in humans and livestock species living in overlapping/adjacent geographical regions

Sphingopyxis Species Isolated from Sand Filter Biofilm at an Australian Drinking Water Treatment Works

Three strains isolated by geosmin enrichment from a sand filter in an Australian drinking water treatment works were genome sequenced to identify their taxonomic placement, and a bench-scale batch experiment confirmed their geosmin-degrading capability. Using the average nucleotide identity based on the MUMmer algorithm (ANIm), pairwise digital DNA-DNA hybridization (dDDH), and phylogenomic analyses, the strains were identified as Sphingopyxis species

Multilocus Genotyping of Human Giardia Isolates Suggests Limited Zoonotic Transmission and Association between Assemblage B and Flatulence in Children

Giardia intestinalis is a protozoan parasite found world-wide and it is a major cause of diarrhea in humans and other mammals. The genetic variability within G. intestinalis is high with eight distinct genotypes or assemblages (A-H). Here we performed sequence-based multilocus genotyping of around 200 human Giardia isolates. We found evidence of limited zoonotic transmission of certain A subtypes and an association between flatulence and assemblage B infection in children. This shows that it is important to investigate different assemblages and sub-assemblages of G. intestinalis in human infections in order to understand the clinical significance, zoonotic potential, sequence divergence, and transmission pathways of this parasite

Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate

<p>Abstract</p> <p>Background</p> <p><it>Giardia intestinalis </it>is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the <it>Giardia intestinalis </it>species, we have performed genome sequencing and analysis of a wild-type <it>Giardia intestinalis </it>sample from the assemblage E group, isolated from a pig.</p> <p>Results</p> <p>We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse <it>Giardia intestinalis </it>isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of <it>Giardia </it>revealed differential rates of divergence among cellular processes.</p> <p>Conclusions</p> <p>Our results indicate that despite a well conserved core of genes there is significant genome variation between <it>Giardia </it>isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the <it>Giardia </it>genomes and enables the identification of functionally important variation.</p

New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species

Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: A systematic analysis for the Global Burden of Disease Study 2013

Background Up-to-date evidence on levels and trends for age-sex-specific all-cause and cause-specific mortality is essential for the formation of global, regional, and national health policies. In the Global Burden of Disease Study 2013 (GBD 2013) we estimated yearly deaths for 188 countries between 1990, and 2013. We used the results to assess whether there is epidemiological convergence across countries. Methods We estimated age-sex-specific all-cause mortality using the GBD 2010 methods with some refinements to improve accuracy applied to an updated database of vital registration, survey, and census data. We generally estimated cause of death as in the GBD 2010. Key improvements included the addition of more recent vital registration data for 72 countries, an updated verbal autopsy literature review, two new and detailed data systems for China, and more detail for Mexico, UK, Turkey, and Russia. We improved statistical models for garbage code redistribution. We used six different modelling strategies across the 240 causes; cause of death ensemble modelling (CODEm) was the dominant strategy for causes with sufficient information. Trends for Alzheimer's disease and other dementias were informed by meta-regression of prevalence studies. For pathogen-specific causes of diarrhoea and lower respiratory infections we used a counterfactual approach. We computed two measures of convergence (inequality) across countries: the average relative difference across all pairs of countries (Gini coefficient) and the average absolute difference across countries. To summarise broad findings, we used multiple decrement life-tables to decompose probabilities of death from birth to exact age 15 years, from exact age 15 years to exact age 50 years, and from exact age 50 years to exact age 75 years, and life expectancy at birth into major causes. For all quantities reported, we computed 95% uncertainty intervals (UIs). We constrained cause-specific fractions within each age-sex-country-year group to sum to all-cause mortality based on draws from the uncertainty distributions. Findings Global life expectancy for both sexes increased from 65·3 years (UI 65·0-65·6) in 1990, to 71·5 years (UI 71·0-71·9) in 2013, while the number of deaths increased from 47·5 million (UI 46·8-48·2) to 54·9 million (UI 53·6-56·3) over the same interval. Global progress masked variation by age and sex: for children, average absolute differences between countries decreased but relative differences increased. For women aged 25-39 years and older than 75 years and for men aged 20-49 years and 65 years and older, both absolute and relative differences increased. Decomposition of global and regional life expectancy showed the prominent role of reductions in age-standardised death rates for cardiovascular diseases and cancers in high-income regions, and reductions in child deaths from diarrhoea, lower respiratory infections, and neonatal causes in low-income regions. HIV/AIDS reduced life expectancy in southern sub-Saharan Africa. For most communicable causes of death both numbers of deaths and age-standardised death rates fell whereas for most non-communicable causes, demographic shifts have increased numbers of deaths but decreased age-standardised death rates. Global deaths from injury increased by 10·7%, from 4·3 million deaths in 1990 to 4·8 million in 2013; but age-standardised rates declined over the same period by 21%. For some causes of more than 100 000 deaths per year in 2013, age-standardised death rates increased between 1990 and 2013, including HIV/AIDS, pancreatic cancer, atrial fibrillation and flutter, drug use disorders, diabetes, chronic kidney disease, and sickle-cell anaemias. Diarrhoeal diseases, lower respiratory infections, neonatal causes, and malaria are still in the top five causes of death in children younger than 5 years. The most important pathogens are rotavirus for diarrhoea and pneumococcus for lower respiratory infections. Country-specific probabilities of death over three phases of life were substantially varied between and within regions. Interpretation For most countries, the general pattern of reductions in age-sex specific mortality has been associated with a progressive shift towards a larger share of the remaining deaths caused by non-communicable disease and injuries. Assessing epidemiological convergence across countries depends on whether an absolute or relative measure of inequality is used. Nevertheless, age-standardised death rates for seven substantial causes are increasing, suggesting the potential for reversals in some countries. Important gaps exist in the empirical data for cause of death estimates for some countries; for example, no national data for India are available for the past decade. Funding Bill &amp; Melinda Gates Foundation