PubMed Health⌕ Search

Biomedical subjects

R M Chalmers

Publications and source records attributed to R M Chalmers.

At least 19 recordsLinked to original sources
PubMed2026

Genome-targeted enrichment and sequencing of human-infecting Cryptosporidium spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (&#x2009;<&#x2009;40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of Cryptosporidium species that have been found to infect humans (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage, and facilitates analyses of genetic variants in many samples, while decreasing overall costs.

Cryptosporidium↗
PubMed2025

GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (<40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of human-infecting Cryptosporidium species ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium spp. DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage and the reliability of detecting species and mixed infections within a sample, and allows assessment of genetic variation via SNP calling, while decreasing costs.

Journal Article↗
PubMed2024

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Journal Article↗
PubMed2024

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

Cryptosporidium↗
PubMed2006

Sample prevalence and molecular characterisation of Cryptosporidium andersoni within a dairy herd in the United Kingdom.

Bovine cryptosporidiosis is usually an acute diarrhoeal disease of young calves caused by Cryptosporidium parvum. However, chronic infection with Cryptosporidium andersoni has been associated with gastritis, reduced milk yield and poor weight gain in adult cattle. Here we describe the first genetic confirmation and characterisation of C. andersoni from cattle in the United Kingdom and its sample prevalence within a dairy herd. Oocysts measured 7.5+/-0.4 microm x 5.5+/-0.4 microm (7.0-8.5 microm x 4.5-6.5 microm) with a length-to-width ratio of 1.37 (1.08-1.60). The within-herd sample prevalence was 16% (95% confidence intervals=10.4-21.6%). Nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequence analysis of the small subunit rDNA was used to confirm the species and characterise the isolates. Due to the lack of overt, acute, clinical symptoms, the incidence, prevalence and importance of this parasite is probably currently underestimated in cattle in the UK. The potential for zoonotic transmission is unknown.

Animals↗
PubMed2006

Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992-2003.

We reviewed the epidemiological and microbiological characteristics of 89 reported outbreaks of waterborne infectious intestinal disease affecting 4321 people in England and Wales over the period 1992-2003. Public water supplies were implicated in 24 outbreaks (27%), private water supplies in 25 (28%), swimming pools in 35 (39%) and other sources in five outbreaks (6%). Cryptosporidium was implicated in 69% of outbreaks, Campylobacter sp. in 14%, Giardia in 2%, E. coli O157 in 3% and Astrovirus in 1%. From 2000, there was a consistent decline in the number of outbreaks of waterborne disease associated with public water supplies. The incidence rate of outbreaks in recipients of private water supplies may be as high as 35 times the rate in those receiving public water supplies (1830 vs. 53 per million population). Private water suppliers need to be aware of the importance of adequate treatment and the prevention of faecal contamination of storage water. Swimming-pool operators need to ensure chlorination and in particular adequate filtration measures are in place.

Communicable Diseases↗
PubMed2005

Borna disease virus and the evidence for human pathogenicity: a systematic review.

BACKGROUND: Borna disease is a neurological viral disease of veterinary importance in central Europe, although Borna Disease virus (BDV) has been reported to be present in animals in most continents. The hypothesis that BDV is associated with human illness is controversial. However, should even a small fraction of mental illness be attributable to infection with BDV, this would be an important finding, not least because illness in that sub-population would, theoretically, be preventable. METHODS: We systematically reviewed the evidence: that BDV infects humans; for the role of BDV in human neuropsychiatric illness; to assess the suitability of currently available laboratory methods for human epidemiological studies. RESULTS: We identified 75 documents published before the end of January 2000, describing 50 human studies for BDV. There were five case studies and 44 (sero)prevalence studies, in a variety of patient groups. Nineteen prevalence studies (43%) investigated seroprevalence, 11 (25%) investigated viral prevalence and 14 (32%) investigated both. Seroprevalence ranged from 0% to 48%, and prevalence of virus or viral footprints from 0% to 82%. DISCUSSION: Although agreed gold standard tests and evidence for test specificity are lacking, there is evidence that humans are exposed to the virus. Further epidemiological studies are required to establish whether there are associations with disease.

Antibodies, Viral↗
PubMed2005

Borna disease virus and mental health: a cross-sectional study.

BACKGROUND: Borna disease is an infectious neurological disease of horses, sheep and possibly other animals. A role for Borna disease virus (BDV) in human neurological and psychiatric illness has been proposed, but this hypothesis remains controversial. AIM: To investigate the epidemiology of BDV in UK farming communities. DESIGN: Retrospective cohort study. METHODS: We measured the seroprevalence of BDV in the PHLS Farm Cohort, a representative sample of those employed in agriculture in the UK, and investigated the clinical significance of our findings by comparing the prevalence of symptoms of neurotic psychopathology in those found seropositive and seronegative. RESULTS: Seroprevalence was 2.3% (95%CI 1.3- 4.0%) in 1994, 3.1% in 1996 (95%CI 1.9-5.0%) and 2.6% in 1999 (95%CI 1.5%-4.6%). Those living or working on livestock farms had higher seroprevalence (2.6%) than those on mixed (2.3%) or arable (1.6%) farms, but this was not statistically significant. Exposure to horses, sheep and cats did not increase risk of seropositivity. Seropositives were no more likely to report symptoms of psychiatric morbidity. DISCUSSION: UK farming populations appear to be exposed to Borna disease virus. However, we found no evidence that exposure to BDV was associated with morbidity in this healthy occupational cohort.

Adult↗
PubMed2003

Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom.

A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. muris was rarely found in wild mammals and C. andersoni oocysts were never detected in livestock. Faecal samples from 3721 individuals gave cumulative 6-year prevalences for C. parvum as follows: bull beef, 3.6%; dairy cows, 3.5%; ewes, 6.4%; horses, 8.9%; calves (home bred), 52%; calves (bought-in) 23.2%; lambs, 12.9%; small wild mammals (rodents) living in and around farm buildings, 32.8%; small wild mammals (mainly rodents) living in areas of pasture, 29.9%. Animal categories with the highest prevalences also shed the highest average oocyst numbers per gram of faeces (ranging from 1.4 x 10(3) for bull beef to 1.1 x 10(5) for calves). Analysis of annual and seasonal data for each animal category revealed that patterns of infection were variable and sporadic; this means that short-term sampling was never likely to provide a true or representative picture. Seasonally combined data for adult livestock, young livestock and small wild mammals showed all three categories tended to have the highest Cryptosporidium prevalences in the autumn. Calves were separated from their dams within 24h of birth and yet showed high prevalence of infection in most years despite the low prevalence for the dairy herd. It is possible the coincidence of high autumn prevalence in mice with the main period for the rearing of calves contributed to the infection of the latter. The farming estate was used to teach students of agriculture and took pride in good land management and husbandry practices that produced well fed and healthy livestock. The data from this estate may represent, therefore, the baseline, the lowest possible levels to be expected, for Cryptosporidium infection and oocyst production on a lowland farm in the United Kingdom.

Animals↗
PubMed2003

Electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported and autochthonous cases of human cryptosporidiosis in the United Kingdom.

Cryptosporidium parvum oocyst DNA samples (n = 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (approximately 300 bp) and second internal transcribed spacer (pITS-2) (approximately 230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of C. parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of approximately 1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C. parvum type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (n = 20; one child nursery outbreak) and type 2 (n = 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of approximately 1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.

Animals↗
PubMed2003

Mental health of British farmers.

AIMS: To estimate the prevalence of neurotic symptoms in a sample of British farmers, to investigate whether farming characteristics are associated with psychiatric morbidity, and to test the hypothesis that British farmers have a higher prevalence of depression and thoughts of life not worth living than the British household population. METHODS: A total of 425 farmers from Hereford, Norwich, and Preston completed the Revised Clinical Interview Schedule (CIS-R) by computer between March and July 1999. The comparison cohort consisted of 9830 private householders aged 16-64 from the Office of Population Censuses and Surveys National Psychiatric Morbidity Surveys of Great Britain carried out in 1993 in which the CIS-R was administered. All analyses used the commands developed specifically for survey data available in Stata version 6.0. RESULTS: Taking a threshold of an overall score of 12 or more on the CIS-R, only 6% of farmers reported clinically relevant psychiatric morbidity. Psychiatric morbidity was not significantly associated with farm type or size in this study. Farmers reported a lower prevalence of psychiatric morbidity than the general population but were more likely to report thinking that life is not worth living, particularly after the low prevalence of psychiatric morbidity had been taken into account (odds ratio 2.56, 95% CI 1.39 to 4.69). When restricting the comparison to only rural or semirural householders, this increased risk was even more pronounced (odds ratio 3.26, 95% CI 1.51 to 7.02). CONCLUSIONS: The relation between depression and suicidal ideation seems to be quite different among farmers and the general population and warrants further investigation. We have shown it is possible to measure mental health systematically in a sample of British farmers. This study should be repeated in the aftermath of the foot and mouth crisis.

Adolescent↗
PubMed2002

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep.

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.

Animals↗
PubMed2002

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. Mutations in the CHAC gene on 9q21 were recently found to cause chorea-acanthocytosis. CHAC encodes a large, novel protein with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in CHAC were screened for mutations by denaturing high-performance liquid chromatography in 43 probands with ChAc. We identified 57 different mutations, 54 of which have not previously been reported, in 39 probands. The novel mutations comprise 15 nonsense, 22 insertion/deletion, 15 splice-site and two missense mutations and are distributed throughout the CHAC gene. Three mutations were found in multiple families within this or our previous study. The preponderance of mutations that are predicted to cause absence of gene product is consistent with the recessive inheritance of this disease. The high proportion of splice-site mutations found is probably a reflection of the large number of exons that comprise the CHAC gene. The CHAC protein product, chorein, appears to have a certain tolerance to amino-acid substitutions since only two out of nine substitutions described here appear to be pathogenic.

Chorea↗
PubMed2002

Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides.

AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.

Animals↗