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

The Eco-Epidemiology of Pacific Coast Tick Fever in California

Rickettsia philipii (type strain “Rickettsia 364D”), the etiologic agent of Pacific Coast tick fever (PCTF), is transmitted to people by the Pacific Coast tick, Dermacentor occidentalis. Following the first confirmed human case of PCTF in 2008, 13 additional human cases have been reported in California, more than half of which were pediatric cases. The most common features of PCTF are the presence of at least one necrotic lesion known as an eschar (100%), fever (85%), and headache (79%); four case-patients required hospitalization and four had multiple eschars. Findings presented here implicate the nymphal or larval stages of D. occidentalis as the primary vectors of R. philipii to people. Peak transmission risk from ticks to people occurs in late summer. Rickettsia philipii DNA was detected in D. occidentalis ticks from 15 of 37 California counties. Similarly, non-pathogenic Rickettsia rhipicephali DNA was detected in D. occidentalis in 29 of 38 counties with an average prevalence of 12.0% in adult ticks. In total, 5,601 ticks tested from 2009 through 2015 yielded an overall R. philipii infection prevalence of 2.1% in adults, 0.9% in nymphs and a minimum infection prevalence of 0.4% in larval pools. Although most human cases of PCTF have been reported from northern California, acarological surveillance suggests that R. philipii may occur throughout the distribution range of D. occidentalis

A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland.

BACKGROUND: This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine beta-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). RESULTS: KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37 degrees C. In contrast, at the permissive temperature of 33 degrees C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37 degrees C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode beta-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37 degrees C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. CONCLUSION: KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland

Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing.

BACKGROUND: Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification. RESULTS: This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples. CONCLUSIONS: The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection

Recruitment of ethnic minority patients to a cardiac rehabilitation trial: the Birmingham Rehabilitation Uptake Maximisation (BRUM) study [ISRCTN72884263]

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Concerns have been raised about low participation rates of people from minority ethnic groups in clinical trials. However, the evidence is unclear as many studies do not report the ethnicity of participants and there is insufficient information about the reasons for ineligibility by ethnic group. Where there are data, there remains the key question as to whether ethnic minorities more likely to be ineligible (e.g. due to language) or decline to participate. We have addressed these questions in relation to the Birmingham Rehabilitation Uptake Maximisation (BRUM) study, a randomized controlled trial (RCT) comparing a home-based with a hospital-based cardiac rehabilitation programme in a multi-ethnic population in the UK. METHODS: Analysis of the ethnicity, age and sex of presenting and recruited subjects for a trial of cardiac rehabilitation in the West-Midlands, UK. PARTICIPANTS: 1997 patients presenting post-myocardial infarction, percutaneous transluminal coronary angioplasty or coronary artery bypass graft surgery. DATA COLLECTED: Exclusion rates, reasons for exclusion and reasons for declining to participate in the trial by ethnic group. RESULTS: Significantly more patients of South Asian ethnicity were excluded (52% of 'South Asian' v 36% 'White European' and 36% 'Other', p < 0.001). This difference in eligibility was primarily due to exclusion on the basis of language (i.e. the inability to speak English or Punjabi). Of those eligible, similar proportions were recruited from the different ethnic groups (white, South Asian and other). There was a marked difference in eligibility between people of Indian, Pakistani or Bangladeshi origin. CONCLUSION: Once eligible for this trial, people from different ethnic groups were recruited in similar proportions. The reason for ineligibility in the BRUM study was the inability to support the range of minority languages.The BRUM study is funded by the NHS HTA Programme

An international comparative study of blood pressure in populations of European vs. African descent

Background: The consistent finding of higher prevalence of hypertension in US blacks compared to whites has led to speculation that African-origin populations are particularly susceptible to this condition. Large surveys now provide new information on this issue. Methods: Using a standardized analysis strategy we examined prevalence estimates for 8 white and 3 black populations (N = 85,000 participants). Results: The range in hypertension prevalence was from 27 to 55% for whites and 14 to 44% for blacks. Conclusions: These data demonstrate that not only is there a wide variation in hypertension prevalence among both racial groups, the rates among blacks are not unusually high when viewed internationally. These data suggest that the impact of environmental factors among both populations may have been under-appreciated

Top and Bottom Seesaw from Supersymmetric Strong Dynamics

We propose a top and bottom seesaw model with partial composite top and bottom quarks. Such composite quarks and topcolor gauge bosons are bound states from supersymmetric strong dynamics by Seiberg duality. Supersymmetry breaking also induces the breaking of topcolor into the QCD gauge coupling. The low energy description of our model reduces to a complete non-minimal extension of the top seesaw model with bottom seesaw. The non-minimal nature is crucial for Higgs mixings and the appearance of light Higgs fields. The Higgs fields are bound states of partial composite particles with the lightest one compatible with a 125 GeV Higgs field which was discovered at the LHC.Comment: Minor changes, Published Versio

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Complement, coagulation and fibrinolysis contribute to the pathology of many respiratory diseases. Here we detail the biphasic activation of these pathways following nasal allergen challenge. Understanding these mechanisms may lead to therapeutic insight in common respiratory diseases

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