Effects of drinking-water filtration on Cryptosporidium seroepidemiology, Scotland.

Continuous exposure to low levels of Cryptosporidium oocysts is associated with production of protective antibodies. We investigated prevalence of antibodies against the 27-kDa Cryptosporidium oocyst antigen among blood donors in 2 areas of Scotland supplied by drinking water from different sources with different filtration standards: Glasgow (not filtered) and Dundee (filtered). During 2006-2009, seroprevalence and risk factor data were collected; this period includes 2007, when enhanced filtration was introduced to the Glasgow supply. A serologic response to the 27-kDa antigen was found for ≈75% of donors in the 2 cohorts combined. Mixed regression modeling indicated a 32% step-change reduction in seroprevalence of antibodies against Cryptosporidium among persons in the Glasgow area, which was associated with introduction of enhanced filtration treatment. Removal of Cryptosporidium oocysts from water reduces the risk for waterborne exposure, sporadic infections, and outbreaks. Paradoxically, however, oocyst removal might lower immunity and increase the risk for infection from other sources.

Cryptic parasite revealed improved prospects for treatment and control of human cryptosporidiosis through advanced technologies.

Cryptosporidium is an important genus of parasitic protozoa of humans and other vertebrates and is a major cause of intestinal disease globally. Unlike many common causes of infectious enteritis, there are no widely available, effective vaccine or drug-based intervention strategies for Cryptosporidium, and control is focused mainly on prevention. This approach is particularly deficient for infections of severely immunocompromised and/or suppressed, the elderly or malnourished people. However, cryptosporidiosis also presents a significant burden on immunocompetent individuals, and can, for example have lasting effects on the physical and mental development of children infected at an early age. In the last few decades, our understanding of Cryptosporidium has expanded significantly in numerous areas, including the parasite life-cycle, the processes of excystation, cellular invasion and reproduction, and the interplay between parasite and host. Nonetheless, despite extensive research, many aspects of the biology of Cryptosporidium remain unknown, and treatment and control are challenging. Here, we review the current state of knowledge of Cryptosporidium, with a focus on major advances arising from the recently completed genome sequences of the two species of greatest relevance in humans, namely Cryptosporidium hominis and Cryptosporidium parvum. In addition, we discuss the potential of next-generation sequencing technologies, new advances in in silico analyses and progress in in vitro culturing systems to bridge these gaps and to lead toward effective treatment and control of cryptosporidiosis.

First genetic classification of Cryptosporidium and Giardia from HIV/AIDS patients in Malaysia.

Given the HIV epidemic in Malaysia, genetic information on opportunistic pathogens, such as Cryptosporidium and Giardia, in HIV/AIDS patients is pivotal to enhance our understanding of epidemiology, patient care, management and disease surveillance. In the present study, 122 faecal samples from HIV/AIDS patients were examined for the presence of Cryptosporidium oocysts and Giardia cysts using a conventional coproscopic approach. Such oocysts and cysts were detected in 22.1% and 5.7% of the 122 faecal samples, respectively. Genomic DNAs from selected samples were tested in a nested-PCR, targeting regions of the small subunit (SSU) of nuclear ribosomal RNA and the 60kDa glycoprotein (gp60) genes (for Cryptosporidium), and the triose-phosphate isomerase (tpi) gene (for Giardia), followed by direct sequencing. The sequencing of amplicons derived from SSU revealed that Cryptosporidium parvum was the most frequently detected species (64% of 25 samples tested), followed by C. hominis (24%), C. meleagridis (8%) and C. felis (4%). Sequencing of a region of gp60 identified C. parvum subgenotype IIdA15G2R1 and C. hominis subgenotypes IaA14R1, IbA10G2R2, IdA15R2, IeA11G2T3R1 and IfA11G1R2. Sequencing of amplicons derived from tpi revealed G. duodenalis assemblage A, which is of zoonotic importance. This is the first report of C. hominis, C. meleagridis and C. felis from Malaysian HIV/AIDS patients. Future work should focus on an extensive analysis of Cryptosporidium and Giardia in such patients as well as in domestic and wild animals, in order to improve the understanding of transmission patterns and dynamics in Malaysia. It would also be particularly interesting to establish the relationship among clinical manifestation, CD4 cell counts and genotypes/subgenotypes of Cryptosporidium and Giardia in HIV/AIDS patients. Such insights would assist in a better management of clinical disease in immuno-deficient patients as well as improved preventive and control strategies.

Identification of a high diversity of Cryptosporidium species genotypes and subtypes in a pediatric population in Nigeria.

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.

Eradication of Blastocystis carriage with antimicrobials: reality or delusion?

Metronidazole constitutes a mainstay in the antimicrobial therapy of intestinal protozoa, and is also traditionally considered first-line therapy in cases where there is a requirement to treat Blastocystis, a common protist of disputable clinical significance. Many compounds have been used in attempts to eradicate the parasite, and an accumulating body of data indicates that successful antimicrobial eradication of Blastocystis is far from straightforward. This review focuses on some issues that prevent us from reaching a clear understanding of how to eradicate Blastocystis based on chemotherapeutic intervention, by focusing on conflicting reports on the efficacy of metronidazole and other compounds and study design and data limitations. The review provides a comprehensive overview of antimicrobials used to target Blastocystis, and discusses issues pertaining to drug resistance, treatment failure, and reinfection. Finally, key methodological and molecular diagnostic tools that will assist in the generation of data required to improve current knowledge are identified and discussed.

Cryptosporidium: detection in water and food.

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.

New tools provide further insights into Giardia and Cryptosporidium biology.

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.

Molecular detection of Giardia contamination in water bodies in a zoo.

We used a combined microscopy-molecular approach to determine the occurrence and identities of waterborne Giardia sp. cysts isolated from 18 separate, 10l grab samples collected from a Malaysian zoo. Microscopy revealed that 17 of 18 samples were Giardia cyst positive with concentrations ranging from 1 to 120 cysts/l. Nine (52.9%) of the 17 cyst positive samples produced amplicons of which 7 (77.8%) could be sequenced. Giardia duodenalis assemblage A (6 of 7) and assemblage B (1 of 7), both infectious to humans, were identified at all sampling sites at the zoo. The presence of human infectious cysts raises public health issues, and their occurrence, abundance and sources should be investigated further. In this zoo setting, our data highlight the importance of incorporating environmental sampling (monitoring) in addition to routine faecal examinations to determine veterinary and public health risks, and water monitoring should be considered for inclusion as a separate element in hazard analysis, as it often has a historical (accumulative) connotation.

Detection and molecular characterization of Giardia isolated from recreational lake water in Malaysia.

Nine 50-l surface water samples from a Malaysian recreational lake were examined microscopically using an immunomagnetisable separation-immunofluorescent method. No Cryptosporidium oocysts were detected, but 77.8% of samples contained low numbers of Giardia cysts (range, 0.17-1.1 cysts/l), which were genetically characterised by SSU rRNA gene sequencing. Genotype analyses indicated the presence of Giardia duodenalis assemblage A suggesting potential risk to public health. The present study represents the first contribution to our knowledge of G. duodenalis assemblages in Malaysian recreational water.

Pursuing the clinical significance of Blastocystis--diagnostic limitations.

The clinical significance of one of the most prevalent single-celled intestinal parasites worldwide, Blastocystis, remains unsettled. A plethora of clinical and epidemiological studies have been undertaken to generate data on its prevalence in different populations and investigate the role of the parasite as a cause of gastro- and extra-intestinal disease. In this article, we pinpoint limitations of studies that seek to determine the clinical significance of Blastocystis, based on shortcomings in our understanding of Blastocystis diagnosis and biology, and identify methodologies for further studies aimed at determining the molecular epidemiology and clinical impact of this parasite.

An outbreak of cryptosporidiosis in a collection of Stone curlews (Burhinus oedicnemus) in Dubai.

We describe an outbreak of cryptosporidiosis in Stone curlews kept in a mixed-species rearing unit in Dubai. Cryptosporidium was the predominant intestinal pathogen detected, although microbiological investigations revealed a concurrent Salmonella infantis infection in two of the 29 Stone curlew chicks that died. Nineteen of 29 birds had catarrhal enteritis associated with histopathological findings of numerous Cryptosporidium developmental stages at the mucosal surface. Catarrhal enteritis was present without associated Cryptosporidium oocysts in five cases. Histology of the intestine, faecal examination by direct microscopy and antigenic detection by immunochromatography revealed the presence of Cryptosporidium spp. associated with catarrhal enteritis in intestinal sections and faeces. Clinical and histopathological outcomes of infection were severe, including disruption of intestinal epithelial integrity, the presence of numerous endogenous Cryptosporidium stages in intestinal epithelia and the excretion of large numbers of sporulated oocysts. The application of polymerase chain reaction and restriction fragment length polymorphism techniques at two 18S rRNA and one Cryptosporidium oocyst wall protein gene locus confirmed the presence of Cryptosporidium parvum DNA in faecal samples.

Application of Toxocara canis excretory-secretory antigens and IgG subclass antibodies (IgG1-4) in serodiagnostic assays of human toxocariasis.

A major problem in the serodiagnosis of human toxocariasis in tropical countries is cross-reaction with antibodies to other helminthic diseases and a lack of sensitivity. The majority of tests currently available use total IgG and, in this study, the use of peroxidase-conjugated anti-human IgG subclass antibodies (IgG1-4) was compared with total IgG for the diagnosis of human toxocariasis by using Toxocara excretory-secretory (TES) antigens in an enzyme-linked immunosorbent assay (ELISA) format. All four IgG subclass antibodies gave approximately 10-fold increases in optical density (OD) values for 50 toxocariasis patients compared to 29 healthy normals; this was significantly greater than the approximate doubling of OD values seen in the total IgG-ELISA format. IgG2 gave by far the greatest sensitivity (values: IgG, 50%; IgG1, 60%; IgG2, 98%; IgG3, 78%; IgG4, 64%). Significant cross-reactivity using all IgG subclasses in the TES ELISA was seen with 141 serum samples from patients with 10 other helminthic infections. However, IgG3 gave the best specificity (values: IgG, 73%; IgG1, 76%; IgG2, 71%; IgG3, 81%; IgG4, 71%). Thus, of the IgG subclass antibodies, IgG2 appeared best and employing this subclass can improve the serodiagnosis of human toxocariasis since it recognises carbohydrate epitopes of TES antigens.

Cryptosporidiosis and filtration of water from Loch Lomond, Scotland.

Previous evidence has suggested an association between consumption of unfiltered water from Loch Lomond, Scotland, and cryptosporidiosis. Before November 1999, this water had been only microstrained and disinfected with chlorine; however, since that time, physical treatment of the water (coagulation, rapid gravity filtration) has been added. To determine risk factors, including drinking water, for cryptosporidiosis, we analyzed data on laboratory-confirmed cases of cryptosporidiosis collected from 1997 through 2003. We identified an association between the incidence of cryptosporidiosis and unfiltered drinking water supplied to the home. The association supports the view that adding a filtration system to minimally treated water can substantially reduce the number of confirmed cryptosporidiosis cases.

The population structure of the Cryptosporidium parvum population in Scotland: a complex picture.

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.

Multilocus analysis of Cryptosporidium hominis and Cryptosporidium parvum isolates from sporadic and outbreak-related human cases and C. parvum isolates from sporadic livestock cases in the United Kingdom.

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.

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.

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.

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.

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.

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.