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Huw V Smith

Publications and source records attributed to Huw V Smith.

9 recordsLinked to original sources
PubMed2006

Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans.

Cryptosporidiosis and giardiasis are major public health concerns. The role of water and food in the epidemiology of these diseases is now well recognized. Molecular techniques are available to determine the species and genotypes of Cryptosporidium and Giardia and to distinguish human from non-human pathogens. Validated methods to determine the species, genotype and subgenotype that are present in heterologous mixtures should be applied to environmental samples to enable the monitoring and characterization of infection sources, disease tracking and the establishment of causative links to both waterborne and foodborne outbreaks. Meaningful interpretation of population structures and occurrence-prevalence baselines can be performed only by analysing a well-planned set of samples from all possible sources taken regularly over time, rather than focusing on outbreak investigations. For food, this includes such analyses in the country of origin.

Animals↗
PubMed2006

Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring.

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.

Animals↗
PubMed2005

A rapid method for extracting oocyst DNA from Cryptosporidium-positive human faeces for outbreak investigations.

We describe a rapid method for extracting and concentrating Cryptosporidium oocysts from human faecal samples with subsequent DNA preparation for mainstream PCR applications. This method consists of extracting faecal lipids using a modified water-ether treatment and releasing DNA from semi-purified oocysts by freeze thawing in lysis buffer. Following immunomagnetisable separation (IMS), recovery rates of 29.5%, 43.2% and 49.8% were obtained from oocyst-negative solid, semi-solid and liquid faeces, respectively, seeded with 100 +/- 2 C. parvum oocysts, which were enumerated by flow cytometry. A retrospective analysis was conducted on 92 positive human faecal samples including 78 oocyst-positive cases from 2 UK cryptosporidiosis outbreaks (outbreak A = 34 samples, outbreak B = 44 samples) and 14 oocyst-positive, sporadic cases. We used primers targeting the Cryptosporidium oocyst wall protein gene (COWP; STN-COWP), the 18S rRNA (direct PCR) and the dihydrofolate reductase gene (dhfr, MAS-PCR) fragments to evaluate extracted DNA by PCR. PCR inhibitors were present in 20 samples when template was co-amplified with the 18S rRNA gene primers and an internal control. Template dilution (1/5) in polyvinylpyrrolidone (10 mg ml(-1), pH 8.0) transformed four PCR-negative samples to PCR-positive and increased amplicon intensity in previously positive samples. Eighteen of 20 PCR-negative samples produced visible amplicons when Taq polymerase concentration in the STN-COWP PCR was increased from 2.5 to 5 U. The STN-COWP PCR assay amplified 90 of 92 samples (97.8%) and the MAS-PCR assay amplified 70 of 92 samples (76.1%) tested. In the absence of inhibitors, DNA equivalent to 3 C. parvum oocysts was amplified.

Animals↗
PubMed2005

Comparison of IgG-ELISA and IgG4-ELISA for Toxocara serodiagnosis.

Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.

Animals↗
PubMed2005

Cryptosporidium excystation and invasion: getting to the guts of the matter.

Cryptosporidium parvum excystation and host cell invasion have been characterized in some detail ultrastructurally. However, until recently, the biochemical and molecular basis of host-parasite interactions and parasite- and host-specific molecules involved in excystation, motility and host cell invasion have been poorly understood. This article describes our understanding of Cryptosporidium excystation and the events leading to host cell invasion, and draws from information available about these processes in other apicomplexans. Many questions remain but, once the specific mechanisms are identified, they could prove to be novel targets for drug delivery.

Animals↗
PubMed2005

Unravelling Cryptosporidium and Giardia epidemiology.

Molecular biology has provided insights into the taxonomy and epidemiology of Cryptosporidium and Giardia, which are major causes of protozoal diarrhoea in humans worldwide. For both genera, previously unrecognized differences in disease, symptomatology, zoonotic potential, risk factors and environmental contamination have been identified using molecular tools that are appropriate for species, genotype and subtype analysis. In this article, to improve understanding of the epidemiology of cryptosporidiosis and giardiasis, we consider specific requirements for the development of more-effective molecular identification and genotyping systems that should be applicable to both clinical and environmental samples.

Animals↗
PubMed2004

New drugs and treatment for cryptosporidiosis.

PURPOSE OF REVIEW: There is a history of inadequate treatments for cryptosporidiosis and a lack of understanding of the species that cause human disease. Against this background, we review the efficacy of antiparasitic agents, particularly nitazoxanide, which has led to increased treatment options, the potential for immunotherapy, and consider the role of highly active antiretroviral therapy in reducing the incidence of this opportunistic infection. RECENT FINDINGS: Nitazoxanide is effective for cryptosporidiosis in immunocompetent and probably immunocompromised patients (with an alteration in the duration of treatment or the dosing regimen). HIV-infected patients on highly active antiretroviral therapy have a dramatically lower incidence of cryptosporidiosis, attributable to the effects of intestinal immune reconstitution as well as the effect on the CD4 cell count. Protease inhibitors have a direct inhibitory effect on Cryptosporidium infection, suggesting a further reason for the reduction in the incidence of cryptosporidiosis and implying a further possible therapeutic modality. SUMMARY: Cryptosporidiosis remains a significant public health threat. Risk avoidance guidance could be viewed in the more relative terms of risk management depending on the degree of immunosuppression. Of established efficacy in immunocompetent patients, nitazoxanide is also useful for immunocompromised patients. Better prevention and treatment options mean that, in the immunocompromised, this disease is now less common. Immune reconstitution is the key to prevention. Further database mining of the Cryptosporidium genome will assist in the discovery of new genes, biochemical pathways and protective antigens that can be targeted to develop novel therapies for cryptosporidiosis.

AIDS-Related Opportunistic Infections↗
PubMed2004

Optimization of DNA extraction and molecular detection of Cryptosporidium oocysts in natural mineral water sources.

The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65 degrees C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65 degrees C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.

Animals↗