[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.
The Lancet Infectious Diseases 09/2014; · 19.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential of capillary electrophoresis (CE)-based DNA fragment analysis to identify mixed infections by Cryptosporidium parvum subpopulations was validated using high-resolution slab-gel electrophoresis. A selection of genomic DNA samples from C. parvum isolates with CE electropherogram profiles indicative of two concurrent alleles at one or more of six mini and microsatellite loci (MSB, MS5, ML1, ML2, TP14, 5B12) were analysed. These loci were PCR-amplified and products separated on precast Spreadex EL600 slab gels. ML1 PCR products differing by as little as 3 bp in length were visible after Spreadex gel electrophoresis and fragments were clearly separated for all but the ML2 and 5B12 loci, which generated stutter bands. No stuttering was seen for the remaining markers, having three or more nucleotide motifs in the repeat region. For each sample, the two bands of interest were excised separately, DNA extracted and re-amplified by PCR. Sequencing of these PCR products revealed the expected sequences for both alleles at most samples, except for the longest ML2 and 5B12 alleles which generated indeterminate sequences. Two novel MS5 alleles were successfully sequenced after PCR re-amplification. These findings demonstrate the utility of high-resolution Spreadex gels for analysing the polymorphism of satellite markers of Cryptosporidium isolates and support the validity of CE as a reliable and sensitive tool for detecting mixed Cryptosporidium subpopulations in a single-host infection.
Parasitology Research 03/2014; 113(5). · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The three protozoan species Cryptosporidium parvum, C. meleagridis and C. hominis (phylum Apicomplexa) are enteric pathogens of humans. The former two species are zoonotic and the latter is thought to infect only humans. To better characterize the structure and transmission of natural and laboratory-propagated isolates, we analyzed a collection of archived human and animal isolates of these three species by deep-sequencing PCR products amplified from a polymorphic sequence on chromosome 1. Thousands of screened 200-nucleotide sequences were analyzed to compare the diversity among samples, to assess the impact of laboratory propagation on population complexity and to identify taxonomically mixed isolates. Contrary to our expectation, repeated propagation in animals did not reduce intra-isolate diversity nor was diversity associated with host species. Significantly, in most samples, sequences characteristic of a different species were identified. The presence of C. hominis alleles in C. parvum and C. meleagridis isolates confirms earlier reports of mixed isolates and raises the possibility that the host range of C. hominis is broader than typically assumed. In a genetically divergent isolate of C. parvum a majority of sequences was found to be recombinant, suggesting that this genotype originated from a C. parvum x C. hominis recombination event.
[Show abstract][Hide abstract] ABSTRACT: We report the first identified outbreak of cryptosporidiosis with Cryptosporidium cuniculus following a water quality incident in Northamptonshire, UK. A standardised, enhanced Cryptosporidium exposure questionnaire was administered to all cases of cryptosporidiosis after the incident. Stool samples, water testing, microscopy slides and rabbit gut contents positive for Cryptosporidium were typed at the Cryptosporidium Reference Unit, Singleton Hospital, Swansea. Twenty-three people were microbiologically linked to the incident although other evidence suggests an excess of 422 cases of cryptosporidiosis above baseline. Most were adult females; unusually for cryptosporidiosis there were no affected children identified under the age of 5 years. Water consumption was possibly higher than in national drinking water consumption patterns. Diarrhoea duration was negatively correlated to distance from the water treatment works where the contamination occurred. Oocyst counts were highest in water storage facilities. This outbreak is the first caused by C. cuniculus infection to have been noted and it has conclusively demonstrated that this species can be a human pathogen. Although symptomatically similar to cryptosporidiosis from C. parvum or C. hominis, this outbreak has revealed some differences, in particular no children under 5 were identified and females were over-represented. These dissimilarities are unexplained although we postulate possible explanations.
Journal of Water and Health 03/2014; 12(1):41-50. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.
[Show abstract][Hide abstract] ABSTRACT: A weighted, multi-attribute approach was used to compare three methods for direct extraction of Giardia duodenalis DNA from 15 microscopy-positive stools: 1) QiaAMP spin-column method for stools including a ten minute incubation at 95°C, 2) Method 1 preceded by five freeze-thaw cycles and 3) bead-beating with guanidine thiocyanate using a FastPrep-28 machine followed by liquid phase silica purification of DNA. Attributes compared included DNA yield measured using a new triose phosphate isomerase (tpi) gene probe-based real-time PCR, also described here. All three methods shared 100% PCR positivity, while the bead-beating method (3) provided the highest G. duodenalis DNA yield (P<0.01). However, when other weighted attributes, including biocontainment, resources and technical requirements, were also considered spin-column extraction with prior freeze-thaw treatment (method 2) was deemed the most desirable and was selected for use. The tpi real-time PCR typing assay was designed to discriminate between the main human infectious assemblages of G. duodenalis (A and B) and was evaluated initially using standard isolates. Validation using microscopy-positive stools from 78 clinical giardiasis cases revealed 100% typability; 20 (26%) samples contained assemblage A, 56 (72%) assemblage B and two (3%) assemblages A and B. While the epidemiological significance of assemblage distribution will be revealed as more isolates are typed and analysed with patient demographic and exposure data, the utility of this assay and its ready application in our laboratory workflow and result turnaround margins is already evident.
Journal of Medical Microbiology 10/2013; · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by C. parvum and C. hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1-10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no cross-reactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4% respectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4-9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18S locus. This qPCR assay should be a valuable tool for the detection and differentiation of C. hominis and C. parvum in both clinical and environmental samples.
[Show abstract][Hide abstract] ABSTRACT: A universal stool extraction method for recovery of nucleic acids from gastrointestinal pathogens was developed to support rapid diagnostics for the London 2012 Olympics. The method involves mechanical disruption (bead-beating) of the stools followed by automated extraction and detection using real time polymerase chain reaction (PCR). This method had been extensively used in the Infectious Intestinal disease (IID2) study for the isolation of nucleic acid from bacteria and parasites (and was critical for the robust recovery of Cryptosporidium spp), but had not been used for enteric viruses. To ensure this method was universally suitable, panels of samples known to contain target bacteria, viruses or parasites were processed in triplicate using the pre-treatment method routinely used for each target and the novel extraction method (bead-beating). The extracts were tested using real-time PCR and the CT (cycle threshold) values were compared. Results from this study showed that bead-beating improved yields for the bacterial and parasitic targets and was not detrimental to the viral targets. The implementation of this universal method should confer cost and time-saving benefits and streamline the processes required for the characterisation of an array of pathogens from faecal samples.
Journal of Medical Microbiology 07/2013; · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Detection of anti-Cryptosporidium immunoglobulin G (IgG) antibodies in human sera has been used to demonstrate population exposure to this gastro-intestinal protozoan parasite. We characterised the dynamics of IgG antibody responses to two Cryptosporidium parvum (IOWA isolate) sporozoite antigens (15/17kDa and 27kDa) using longitudinal sera taken from laboratory-confirmed cryptosporidiosis cases in England and Wales. The effect of the infecting Cryptosporidium species was also investigated. METHODS: A mini-gel Western blot was used to test sera from ten Cryptosporidium stool-positive diarrhoea patients, taken soon after diagnosis and at 3 month intervals. RESULTS: Overall responses to the 15/17kDa antigen complex were stronger and over a greater range than those to the 27kDa antigen, but declined between 181 and 240 days and were barely detectable thereafter. Responses to the 27kDa antigen were much weaker but remained detectable for a greater length of time. No differences were detected in either antibody response to infection with C. hominis or C. parvum. CONCLUSIONS: The assay appears to be applicable for the study of recent exposure to C. parvum or C. hominis in the United Kingdom population, with strong responses to the 15/17kDa antigen occurring within 6 months of infection.
The Journal of infection 05/2013; · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protozoan Cryptosporidium is a major public and animal health concern. Young children, immunocompromised people, and pre-weaning animals are especially vulnerable, but treatment options are limited and there is no vaccine. A laboratory diagnosis is required to confirm cases of cryptosporidiosis, and species and genotype determination is essential in distinguishing human from non-human sources, understanding transmission, and strengthening the epidemiological evidence for causative links in outbreaks. However, testing is not consistent, as demonstrated by investigation of a significant increase in cases in some European countries during 2012. Many methods employed are laborious and time-consuming; recent advances, translated into diagnostic assays, can improve testing and facilitate typing to support clinical and environmental investigations.
Trends in Parasitology 04/2013; · 6.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidiosis is predominantly caused by two closely related species of protozoan parasites the zoonotic and anthroponotic which diverge phenotypically in respect to host range and virulence. Using comparative genomics we identified two genes displaying overt heterogeneity between species. Although initial work suggested both were species specific, for and for , subsequent study identified an abridged ortholog of in . and showed limited, but significant, similarity to each other and share common features: (i) telomeric location: is the last gene on chromosome 2, whilst is the first gene on chromosome 5, (ii) encode circa 50-kDa secreted proteins with isoelectric points above 10, (iii) are serine rich, and (iv) contain internal nucleotide repeats. Importantly, sequence contains specific SNPs with good discriminatory power useful epidemiologically. -infected patient sera recognized a 50-kDa protein in antigen preparations of but not , consistent with Cops-1 being antigenic for patients. Interestingly, anti-Cops-1 monoclonal antibody (9E1) stained oocyst content and sporozoite surface of only. This study provides a new example of protozoan telomeres as rapidly evolving contingency loci encoding putative virulence factors.
[Show abstract][Hide abstract] ABSTRACT: Starting August 2012, an increase in Cryptosporidium infections was reported in the Netherlands, the United Kingdom and Germany. It represented a 1.8 to 4.9-fold increase compared to previous years. Most samples were C. hominis IbA10G2. A case–control study was performed in the Netherlands but did not identify an endemic source. A case–case study in the north of England found travel abroad to be the most common risk factor.
Euro surveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 01/2013; 18(2). · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Cryptosporidium is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Several studies have reported different degrees of pathogenicity and virulence among Cryptosporidium species and isolates of the same species as well as evidence of variation in host susceptibility to infection. The identification and validation of Cryptosporidium virulence factors have been hindered by the renowned difficulties pertaining to the in vitro culture and genetic manipulation of this parasite. Nevertheless, substantial progress has been made in identifying putative virulence factors for Cryptosporidium. This progress has been accelerated since the publication of the Cryptosporidium parvum and C. hominis genomes, with the characterization of over 25 putative virulence factors identified by using a variety of immunological and molecular techniques and which are proposed to be involved in aspects of host-pathogen interactions from adhesion and locomotion to invasion and proliferation. Progress has also been made in the contribution of host factors that are associated with variations in both the severity and risk of infection. Here we provide a review comprised of the current state of knowledge on Cryptosporidium infectivity, pathogenesis, and transmissibility in light of our contemporary understanding of microbial virulence.
[Show abstract][Hide abstract] ABSTRACT: Water is the most commonly reported vehicle of transmission in Cryptosporidium outbreaks. While mains drinking water quality is highly regulated in industrialised countries, treated recreational water venues remain highly variable and these have emerged as important settings in the transmission of cryptosporidiosis. Epidemiological investigations of outbreaks benefit from supplementary microbiological evidence and, more recently, the application of molecular typing data to link isolates from cases to each other and to suspected sources. This article documents how waterborne Cryptosporidium outbreaks are identified and reported, how such outbreaks have acted as drivers of regulatory change, and some of the recent developments in the detection and investigation of these outbreaks and their spread, especially the application of molecular typing assays.
[Show abstract][Hide abstract] ABSTRACT: Most waterborne outbreaks of cryptosporidiosis are reported from the USA, and in Europe from the UK. However, since 2000 reports of foodborne cryptosporidiosis seem to be skewed towards Nordic countries, although consumers in these countries are apparently less concerned about microbiological contamination of food than consumers elsewhere. Possible reasons for this unexpected geographical distribution might include prolonged survival of oocysts in the Nordic climate, greater exposure due to elevated consumption of higher-risk products (possibly including imported foods), and better outbreak investigation and reporting. Although the risk of foodborne cryptosporidiosis is probably underestimated globally, we suggest that the next outbreak is no more likely to be in a Nordic country than anywhere else.
Trends in Parasitology 11/2012; · 6.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of high resolution molecular tools to study Cryptosporidium parvum and Cryptosporidium hominis intra-species variation is becoming common practice, but there is currently no consensus in the methods used. The most commonly applied tool is partial gp60 gene sequence analysis. However, multi-locus schemes are acknowledged to improve resolution over analysis of a single locus, which neglects potential re-assortment of genes during the sexual phase of the Cryptosporidium life-cycle. Multi-locus markers have been investigated in isolates from a variety of sampling frames, in varying combinations and using different assays and methods of analysis. To identify the most informative markers as candidates for the development of a standardised multi-locus fragment size-based typing (MLFT) scheme to integrate with epidemiological analyses, we examined the published literature. A total of 31 MLFT studies were found, employing 55 markers of which 45 were applied to both C. parvum and C. hominis. Of the studies, 11 had sufficient raw data, from three or more markers, and a sampling frame containing at least 50 samples, for meaningful in-depth analysis using assessment criteria based on the sampling frame, study size, number of markers investigated in each study, marker characteristics (>2 nucleotide repeats) and the combinations of markers generating all possible multi-locus genotypes. Markers investigated differed between C. hominis and C. parvum. When each scheme was analysed for the fewest markers required to identify 95% of all MLFTs, some redundancy was identified in all schemes; an average redundancy of 40% for C. hominis and 27% for C. parvum. Ranking markers, based on the most productive combinations, identified two different sets of potentially most informative candidate markers, one for each species. These will be subjected to technical evaluation including typability (percentage of samples generating a complete multi-locus type) and discriminatory power by direct fragment size analysis and analysed for correlation with epidemiological data in suitable sampling frames. The establishment of a group of users and agreed subtyping scheme for improved epidemiological and public health investigations of C. parvum and C. hominis will facilitate further developments and consideration of technological advances in a harmonised manner.
[Show abstract][Hide abstract] ABSTRACT: A novel Cryptosporidium genotype was identified, among travellers with gastro-intestinal symptoms returning to Great Britain from the Indian subcontinent, for which we propose the name Cryptosporidium viatorum n. sp. The epidemiology of these cases was distinctly different from those with Cryptosporidium parvum and Cryptosporidium hominis. Of the 10 cases identified involving C. viatorum, most were in the first quarter of the year. One occurred in 2007, one in 2008, three in 2010 and five to end March 2011. The median age was 19 years but most were in the 20-29 years age group and seven were male. The symptoms included diarrhoea, abdominal pain, nausea, vomiting and fever. Compared with cases due to C. hominis and C. parvum, vomiting was reported less often, although the duration of gastro-intestinal symptoms was longer. The cases of C. viatorum were all travellers to the Indian subcontinent, whereas cases of C. hominis and C. parvum were more likely to have travelled elsewhere. Cryptosporidium viatorum isolates had indistinguishable sequences at each of the 70 kDa heat shock protein (HSP70), actin and ssrRNA loci which did not match any published previously and, although phylogenetically most similar to Cryptosporidium fayeri, they were distinct (<98% similarity) at the ssrRNA, HSP70 and actin genes. Morphologically, oocysts were typical of predominantly human-infecting species. Cryptosporidium viatorum n. sp. is proposed and work is warranted to investigate further the public health significance and occurrence elsewhere of this emerging parasite.
International journal for parasitology 05/2012; 42(7):675-82. · 3.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium cuniculus was originally detected in rabbits and has been identified as an emerging human pathogen, but the occurrence, prevalence, and epidemiology in human and rabbit populations are poorly understood. As identification of C. cuniculus can be time-consuming and costly using existing molecular assays, a real-time polymerase chain reaction (PCR)-based method targeting specific markers for this species was developed. The assay is based on amplification of the C. cuniculus-specific 60-kDa glycoprotein (GP60) gene using two PCRs targeting subtype families Va and Vb. PCR product formation was monitored by SYBR Green I fluorescence measurement followed by post-amplification melt curve analysis; high resolution melt curve analysis was found to give increased sensitivity over standard melt curve analysis. The real-time PCR correctly identified all 41 C. cuniculus isolates (40 from humans, one from a rabbit) tested, with subtype family in agreement with GP60 gene sequencing. Specificity was demonstrated by lack of detection of nine other Cryptosporidium species and genotypes, including 88 isolates of the closely related species, Cryptosporidium hominis. The PCRs were performed in separate tubes to maximize the possibility of detecting mixed Va-Vb infections; however, none were detected. The potential for multiplexing the reactions was also demonstrated, furthering the utility of the assay for large-scale occurrence and prevalence studies.
Parasitology Research 03/2012; 111(3):1385-90. · 2.33 Impact Factor