Sporadic Cryptosporidium infection in Nigerian children: risk factors with species identification.

A cross-sectional study was conducted to investigate risk factors for sporadic Cryptosporidium infection in a paediatric population in Nigeria. Of 692 children, 134 (19·4%) were infected with Cryptosporidium oocysts. Cryptosporidium spp. were identified in 49 positive samples using PCR-restriction fragment length polymorphism and direct sequencing of the glycoprotein60 (GP60) gene. Generalized linear mixed-effects models were used to identify risk factors for all Cryptosporidium infections, as well as for C. hominis and C. parvum both together and separately. Risk factors identified for all Cryptosporidium infections included malaria infection and a lack of Ascaris infection. For C. hominis infections, stunting and younger age were highlighted as risk factors, while stunting and malaria infection were identified as risk factors for C. parvum infection.

Identification of Cryptosporidium species and genotypes in Scottish raw and drinking waters during a one-year monitoring period.

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).

Cryptosporidium oocysts on fresh produce from areas of high livestock production in Poland.

Samples of fresh vegetables and soft fruit were collected from farmers' markets in the Lublin Area of Poland during 2006-2007; the produce was grown in areas of high to moderate livestock production. Cryptosporidium sp. oocysts were eluted from food surfaces, separated from residual food materials by IMS and identified by immunofluorescence and Nomarski differential interference contrast microscopy. Cryptosporidium sp. oocysts were detected in 6 of 128 vegetable samples (range 1-47 oocysts), but not in any of 35 fruit samples. Both empty and intact oocysts were detected. Species identity of oocyst-positive samples was performed by molecular analysis at four genetic loci. One of two 18S rRNA loci amplified DNA from 5 of the 6 oocyst-positive samples, but insufficient DNA for RFLP or sequencing analysis was available from 4 of these samples. An oocyst-positive celery sample generated an RFLP pattern consistent with C. parvum at two loci, but insufficient DNA was available for subtyping (GP60 sequencing) this isolate. Oocyst-contaminated foods originated from districts with the highest numbers of homesteads possessing cattle herds and no contaminated produce was detected from districts containing lower numbers of cattle-owning homesteads, strengthening the assumption that the origin of the contamination was livestock. The results of this study strengthen the evidence for the potential for zoonotic foodborne transmission of Cryptosporidium.

Development of a method for detection of Giardia duodenalis cysts on lettuce and for simultaneous analysis of salad products for the presence of Giardia cysts and Cryptosporidium oocysts.

We report a method for detecting Giardia duodenalis cysts on lettuce, which we subsequently use to examine salad products for the presence of Giardia cysts and Cryptosporidium oocysts. The method is based on four basic steps: extraction of cysts from the foodstuffs, concentration of the extract and separation of the cysts from food materials, staining of the cysts to allow their visualization, and identification of cysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Cyst staining is also performed using proprietary reagents. The method recovered 46.0% +/- 19.0% (n = 30) of artificially contaminating cysts in 30 g of lettuce. We tested the method on a variety of commercially available natural foods, which we also seeded with a commercially available internal control, immediately prior to concentration of the extract. Recoveries of the Texas Red-stained Giardia cyst and Cryptosporidium oocyst internal controls were 36.5% +/- 14.3% and 36.2% +/- 19.7% (n = 20), respectively. One natural food sample of organic watercress, spinach, and rocket salad contained one Giardia cyst 50 g(-1) of sample as an indigenous surface contaminant.

Cryptosporidium and Giardia as foodborne zoonoses.

Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.

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.

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.

Zoonotic protozoa--food for thought.

Outbreaks of water- and foodborne diseases caused by Cryptosporidium, Giardia and Toxoplasma are well documented. Three features of these zoonotic protozoa ensure a high level of environmental contamination and enhance the likelihood of waterborne transmission. Firstly, they are responsible for disease in a broad range of hosts including man, have a low infectious dose enhancing the possibility of zoonotic transmission, secondly, their transmissive stages are small in size and environmentally robust and thirdly are insensitive to the disinfectants commonly used in the water industry. In addition, there is growing evidence for the role that water and food can play in the transmission of the microsporidia, Balantidium and Blastocystis to humans.

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.

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.

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.

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.

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.

Significance of enhanced morphological detection of Cryptosporidium sp. oocysts in water concentrates determined by using 4',6'-diamidino-2-phenylindole and immunofluorescence microscopy.

Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.