[show abstract][hide abstract] ABSTRACT: A longitudinal study was conducted to determine the epidemiology of Cryptosporidium in 1,636 children in Nigeria. Oocyst prevalence ranged from 15.6% to 19.6% over one year. Cryptosporidium hominis (34), C. parvum (25), C. parvum/C. hominis (4), C. meleagridis (5), Cryptosporidium rabbit genotype (5), Cryptosporidium cervine genotype (3), and C. canis (1) were identified by polymerase chain reaction-restriction fragment length polymorphism analysis. Glycoprotein 60 subgenotyping showed that 28 amplifiable C. hominis isolates consisted of 12 subtypes that belonged to 5 subtype families (Ia, Ib, Id, Ie, and 1 novel subtype family, Ih) and 23 amplifiable C. parvum isolates consisted of 6 subtypes that belonged to 4 subtype families (IIa, IIc, Iii, and IIm). Three C. meleagridis isolates sub-genotyped by sequence analysis of the small subunit ribosomal RNA gene fragment were type 1. This study is the first one to genetically characterize Cryptosporidium species and subtypes in Nigeria and highlights the presence of a high Cryptosporidium diversity in this pediatric population.
The American journal of tropical medicine and hygiene 04/2010; 82(4):608-13. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ubiquity and importance of Giardia and Cryptosporidium as pathogens are reflected in the increasing number of publications concerning these organisms, but they are not the only reason why researchers are increasingly turning their attention to studying Giardia and Cryptosporidium. As new tools and databases become available, it is now possible to investigate fundamental issues related to their biology and relationship with their hosts. In this article, we highlight recent advances in research and outline questions arising that need to be addressed as a way of focusing the attention of the research and health communities and encouraging further dialogue and collaboration.
Trends in Parasitology 10/2009; 25(9):410-6. · 5.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: Water and food are major environmental transmission routes for Cryptosporidium, but our ability to identify the spectrum of oocyst contributions in current performance-based methods is limited. Determining risks in water and foodstuffs, and the importance of zoonotic transmission, requires the use of molecular methods, which add value to performance-based morphologic methods. Multi-locus approaches increase the accuracy of identification, as many signatures detected in water originate from species/genotypes that are not infectious to humans. Method optimisation is necessary for detecting small numbers of oocysts in environmental samples consistently, and further work is required to (i) optimise IMS recovery efficiency, (ii) quality assure performance-based methods, (iii) maximise DNA extraction and purification, (iv) adopt standardised and validated loci and primers, (v) determine the species and subspecies range in samples containing mixtures, and standardising storage and transport matrices for validating genetic loci, primer sets and DNA sequences.
[show abstract][hide abstract] ABSTRACT: We genotyped 297 Scottish C. parvum samples using micro- and minisatellites. Treated as a single population, the population structure was epidemic. When regional populations were analysed, there was evidence of sub-population structure variations. This was dependent upon excluding sub-groups exhibiting significant genetic distance from the main population, implying genetic sub-structuring. We tested the hypothesis that these sub-groups originated outside the UK and demonstrated that one sub-group clustered with Peruvian samples. A geographically comprehensive panel of isolates would fully confirm this result. These data indicate limited sub-structuring within a small geographical area, but substantial sub-structuring over larger geographical distances. Host movement influences parasite diversity and population structure, evidenced by strong correlation (r(2) = 0.9686) between cattle movements and parasite diversity. Thus, the population structure of C. parvum is complex, with sub-populations differing in structure and being influenced by host movements, including the introduction of novel multilocus genotypes from geographically distinct regions.
Infection Genetics and Evolution 04/2008; 8(2):121-9. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Seven species of the intracellular, protozoan parasite, Cryptosporidium, cause diarrhea in humans. Cryptosporidium completes its life cycle in individual hosts, and its infectious dose is small (ID50 = 9-1042 oocysts). Its virulence and pathogenicity are poorly understood. The drug of choice for immunocompetent individuals
is nitazoxanide and modifications of treatment regimens may also increase its usefulness for immunocompromised individuals.
Immune reconstitution using highly active antiretroviral therapy and secondary prophylaxis should be considered in HIV-infected
individuals. Transmission to humans can occur via any mechanism by which material contaminated with feces containing infectious
oocysts from infected human beings or non-human hosts can be swallowed by a susceptible host. Biotic reservoirs include all
potential hosts of human-infectious Cryptosporidium species, while abiotic reservoirs include all vehicles that contain sufficient infectious oocysts to cause human infection,
the most commonly recognized being food and water. Both foodborne and waterborne outbreaks have been documented. In three
out of the six foodborne outbreaks documented, contaminated foodstuffs were implicated as the vehicles of transmission, but
in another two, foodhandlers, rather than indigenous contamination of foodstuff, were implicated in disease transmission.
Increased global sourcing and rapid transport of soft fruit, salad vegetables, and seafood can enhance both the likelihood
of oocyst contamination and oocyst survival. Standardized methods for detecting oocysts on foods must be maximized as there
is no method to augment parasite numbers prior to detection. Oocyst contamination of food can be on the surface of, or in,
the food matrix and products at greatest risk of transmitting infection include those that receive no, or minimal, heat treatment
after they become contaminated. Heating at ≥64.2 for 2 min and exposure to UV light ablates Cryptosporidium parvum infectivity for neonatal mice, while drying/desiccation for 4 h or exposure to 0.03% H2O2 for ≥2 h ablates oocyst viability. Disinfectants and other treatment processes used in the food industry may be detrimental
to oocyst survival or lethal, but further research in this important area is required.
Both foodborne and waterborne outbreaks have been documented. In three out of the six foodborne outbreaks documented, contaminated
foodstuffs were implicated as the vehicles of transmission, but in another two, foodhandlers, rather than indigenous contamination
of foodstuff, were implicated in disease transmission. Increased global sourcing and rapid transport of soft fruit, salad
vegetables, and seafood can enhance both the likelihood of oocyst contamination and oocyst survival. Standardized methods
for detecting oocysts on foods must be maximized as there is no method to augment parasite numbers prior to detection. Oocyst
contamination of food can be on the surface of, or in, the food matrix and products at greatest risk of transmitting infection
include those that receive no, or minimal, heat treatment after they become contaminated. Heating at ≥64.2 for 2 min and exposure
to UV light ablates Cryptosporidium parvum infectivity for neonatal mice, while drying/desiccation for 4 h or exposure to 0.03% H2O2 for ≥2 h ablates oocyst viability. Disinfectants and other treatment processes used in the food industry may be detrimental
to oocyst survival or lethal, but further research in this important area is required.
[show abstract][hide abstract] ABSTRACT: Cryptosporidium parvum and Cryptosporidium hominis isolates from sporadic, drinking water-associated, and intrafamilial human cases together with C. parvum isolates from sporadic cases in livestock were collected in the United Kingdom between 1995 and 1999. The isolates were characterized by analysis of three microsatellite markers (ML1, GP15, and MS5) using PCR amplification. Within C. hominis, four alleles were detected within the GP15 and MS5 loci, and a single type was detected with ML1. C. parvum was more polymorphic; 12 alleles were detected with GP15, 6 were detected with MS5, and 3 were detected with ML1. Multilocus analysis of polymorphisms within the three microsatellite loci was combined with those reported previously for an extrachromosomal small double-stranded RNA. Forty multilocus types were detected within these two species: 9 were detected in C. hominis, and 31 were detected in C. parvum. In C. hominis, heterogeneity was almost exclusively found in samples from sporadic cases. Similarity analysis identified three main groups within C. parvum, and the group that predominated in human infection was also found in livestock. Multilocus types of C. parvum previously identified only in humans were not detected in livestock. Isolates of both C. hominis and C. parvum from separate waterborne outbreaks were genetically homogeneous, suggesting preferential or point source transmission of certain types of these two species of parasites.
Journal of Clinical Microbiology 11/2007; 45(10):3286-94. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 09/2006; 72(8):5428-35. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Microbiological Methods 07/2006; 65(3):512-24. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Trends in Parasitology 05/2006; 22(4):160-7. · 5.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Trends in Parasitology 10/2005; 21(9):430-7. · 5.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Trends in Parasitology 04/2005; 21(3):133-42. · 5.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
Current Opinion in Infectious Diseases 01/2005; 17(6):557-64. · 4.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of food protection 04/2004; 67(3):524-32. · 1.83 Impact Factor