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H V Smith

Publications and source records attributed to H V Smith.

At least 19 recordsLinked to original sources
PubMed2007

Incidence of cryptosporidiosis species in paediatric patients in Malawi.

We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR-RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.

Animals↗
PubMed2006

Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ x cm-2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x cm-2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x cm-2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

Animals↗
PubMed2006

Cryptosporidium parvum sporozoites contain glutathione.

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean +/- S.D. 35 +/- 3.7) compared with controls (3.3 +/- 1.2, P = 0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4'6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm(-2) irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

Animals↗
PubMed2006

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts.

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

Adolescent↗
PubMed2006

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 2: validation.

We report the results of interlaboratory collaborative trials of methods to detect oocysts of the protozoan parasite Cryptosporidium parvum on lettuce and raspberries. The trials involved eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce, and 60 g raspberries. Lettuce samples were artificially contaminated at three levels: low (8.5-14.2 oocysts), medium (53.5-62.6 oocysts), and high (111.3-135.0 oocysts). Non-contaminated lettuce samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated lettuce samples) of 89.6%, and a specificity (correct identification of non-contaminated samples) of 85.4%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 30.4%. The method was just as reproducible between laboratories, as repeatable within a laboratory. Raspberry samples were artificially contaminated at three levels: low (8.5-26.8 oocysts), medium (29.7-65.7 oocysts), and high (53.9-131.3 oocysts). Non-contaminated raspberry samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated raspberry samples) of 95.8%, and a specificity (correct identification of non-contaminated samples) of 83.3%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 44.3%. The method was just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that these assays can be used effectively in analytical microbiological laboratories.

Animals↗
PubMed2006

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods.

No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.

Animals↗
PubMed2006

Fertilization state of Ascaris suum determined by electrorotation.

Electrorotation is a non-invasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. The first detailed electrorotation study of the egg (ovum) of a parasitic nematode, namely Ascaris suum is described to show that electrorotation can rapidly differentiate between fertilized and non-fertilized eggs. Support for this conclusion is by optical microscopy of egg morphology, and also from modelling of the electrorotational response. Modelling was used to determine differences in the dielectric properties of the unfertilized and fertilized eggs, and also to investigate specific differences in the spectra of fertilized eggs only, potentially reflecting embryogenesis. The potential of electrorotation as an investigative tool is shown, as undamaged eggs can be subjected to further non-destructive and destructive techniques, which could provide further insight into parasite biology and epidemiology.

Animals↗
PubMed2005

Viable blastocystis cysts in Scottish and Malaysian sewage samples.

Blastocystis cysts were detected in 38% (47/123) (37 Scottish, 17 Malaysian) of sewage treatment works. Fifty percent of influents (29% Scottish, 76% Malaysian) and 28% of effluents (9% Scottish, 60% Malaysian) contained viable cysts. Viable cysts, discharged in effluent, provide further evidence for the potential for waterborne transmission of Blastocystis.

Animals↗
PubMed2005

Spironucleosis (Hexamitiasis, Hexamitosis) in the ring-necked pheasant (Phasianus colchicus): detection of cysts and description of Spironucleus meleagridis in stained smears.

Trophozoites and cysts of Spironucleus (Hexamita) meleagridis were detected in the intestinal fluid and mucus of pheasant poults with spironudeosis (hexamitiasis, hexamitosis) following staining with Heidenhain iron hematoxylin (HIH) and the Romanowsky-type stain Hemacolor. Their morphology was consistent with that of flagellates of the genus Spironucleus, and bright-field morphologic observations were confirmed by transmission electron microscopy. Cysts occurred mostly within intestinal mucus, which was firmly compressed between microscope slides prior to staining. The internal structures of cysts were similar to those of trophozoites, allowing them to be confidently recognized. Hemacolor provided differential color staining of trophozoites and cysts, allowing accurate identification of S. meleagridis life cycle stages, even in smears in which there was heavy background staining. While HIH often produced dearer and more detailed staining of protozoan structures, in the context of a diagnostic laboratory its use was outweighed by the ease of use, rapidity of results, and differential color staining provided by Hemacolor. The possible significance of a resistant cystic stage in the life cyde of S. meleagridis is discussed.

Animals↗
PubMed2004

Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.

Amino Acid Sequence↗
PubMed2004

Analysis of parasites by electrorotation.

AIMS: The application of the AC electrokinetic technique of electrorotation for studying eukaryotic parasite transmission stages is reviewed. Electrorotation is a noninvasive technique that utilizes electrically energized microelectrode structures within micro-fluidic chambers to probe the physiological structure of micro-organisms. Application of the technique to the transmission life cycle stages of three separate genera of protozoan parasites, Cryptosporidium, Giardia and Cyclospora, and one nematode genus Ascaris, each of significant public health importance, is described. METHODS AND RESULTS: Standard electrorotation apparatus, consisting of micro-fabricated electrodes in a fluidic chip, quadrature sinusoidal signal generator, microscope and image capture system, was used to study each organism. Spectra of cellular rotation rate were recorded as a function of applied electric field frequency and compared with standardized biological tests, where appropriate, to illustrate the effectiveness and versatility of the electrorotation technique. CONCLUSIONS: Electrorotational determination of the viability of individual G. intestinalis cysts, Cryptosporidium parvum and Cyclospora cayetanensis oocysts has been achieved. The sporulation state of Cyclospora cayetanensis oocysts was also readily determined, as was the fertilization state of A. suum ova. SIGNIFICANCE AND IMPACT OF THE STUDY: Electrorotation is a simple, noninvasive and versatile analytical technique suited to a wide range of particle types and capable of incorporation into integrated Lab-on-a-chip devices.

Animals↗
PubMed2004

Survival of Cryptosporidium parvum oocysts after prolonged exposure to still natural mineral waters.

The survival kinetics of purified Cryptosporidium parvum oocysts of both human and ovine origin, immersed in four still natural mineral waters (total dissolved salts ranging from 91 mg/liter to 430 mg/liter) and reverse osmosis water was assessed by inclusion or exclusion of the fluorogenic vital dyes 4',6-diamidino-2-phenylindole and propidium iodide over a 12-week period. Semipermeable chambers were used to contain the oocysts while immersed in each mineral water type, permitting both intimate interactions between oocysts and matrices and straightforward sampling for viability assessments. The viability of both oocyst types, assessed at weekly intervals, remained unaltered after 12 weeks at 4 degrees C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20 degrees C. At 20 degrees C, approximately 30% of oocysts remained viable after 12 weeks incubation. Here, temperature was the major factor that adversely affected oocyst survival, although higher mineral content was also proportionally and significantly associated with this increased oocyst inactivation. The prolonged survival of oocysts at 4 degrees C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature approximately 10 degrees C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.

Animals↗
PubMed2004

Invasive group A streptococcal disease in North Queensland (1996 - 2001).

BACKGROUND & OBJECTIVES: The incidence of group A streptococcal (GAS) invasive infections have been increasing worldwide. The aim of this study was to characterize clinical and microbiological features of isolates obtained from invasive GAS infections in North Queensland, Australia between 1996 and 2001. METHODS: Clinical and demographic data were collected prospectively. Isolates were biotyped, emm sequenced, M typed and tested for antibiotic sensitivity using E-test. Detection of the presence of the streptococcal pyrogenic exotoxin (spe) and fibronectin binding protein (prtF1) genes was also carried out. RESULTS: There were 109 isolates from blood and sterile sites. All isolates were sensitive to penicillin. Tetracycline and erythromycin resistance was seen in 11 and 2.7 per cent of isolates respectively. The isolates were evenly distributed by age and sex. The overall mortality was 7 per cent and there were 18 cases of streptococcal toxic shock syndrome (STSS) in which the mortality was 22 per cent. Indigenous patients had a crude incidence rate of 82.5 per 100,000 per year compared with 10.3 per 100,000 per year in the non-indigenous patients. There was no predominance of emm / M type or association of spe type with STSS. There was also no relationship between the presence of the prtF1 gene and invasive disease. INTERPRETATION & CONCLUSION: Invasive group A streptococci from North Queensland are similar to those from the Northern Territory of Australia in that no single strain is predominant. The indigenous population is overrepresented. Invasiveness and the development of streptococcal toxic shock is not related to the presence of the prtF1 gene or spe a or c.

Adhesins, Bacterial↗
PubMed2003

Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay.

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

Animals↗
PubMed2003

Burkholderia pseudomallei: another emerging pathogen in cystic fibrosis.

BACKGROUND: Burkholderia pseudomallei is an important cause of acute fulminant pneumonia and septicaemia in tropical regions of northern Australia and south east Asia. Subacute and chronic forms of the disease also occur. There have been three recent reports of adults with cystic fibrosis (CF) who presumably acquired B pseudomallei infection during extended vacations or residence in either Thailand or northern Australia. METHODS: The clinical course, molecular characteristics, serology and response to treatment are described in four adult CF patients infected with B pseudomallei. Polymerase chain reaction (PCR) based methods were used to confirm B pseudomallei and exclude B cepacia complex. Genotyping was performed using randomly amplified polymorphic DNA (RAPD) PCR and pulsed field gel electrophoresis (PFGE). RESULTS: Four patients are described with a mean duration of infection of 32 months. All but one patient lived in tropical Queensland. Two patients (with the longest duration of infection) deteriorated clinically and one subsequently died of respiratory failure. Both responded to intravenous treatment specifically targeting B pseudomallei. Another patient suffered two severe episodes of acute bronchopneumonia following acquisition of B pseudomallei. Eradication of the organism was not possible in any of the cases. PFGE of a sample isolate from each patient revealed the strains to be unique and RAPD analysis showed retention of the same strain within an individual over time. CONCLUSIONS: These findings support a potential pathogenic role for B pseudomallei in CF lung disease, producing both chronic infection and possibly acute bronchopneumonia. Identical isolates are retained over time and are unique, consistent with likely environmental acquisition and not person to person spread. B pseudomallei is emerging as a significant pathogen for patients with CF residing and holidaying in the tropics.

Adolescent↗