[Show abstract][Hide abstract] ABSTRACT: During weeks 32-33, 2013, 24 cases of cryptosporidiosis were notified in the city of Halle (annual mean 2008-2012: 9 cases). We investigated the outbreak to identify the source and recommend control measures, considering that between weeks 23-25 the river Saale which flows through the city centre overflowed the floodplain, parts of the city centre and damaged sewage systems.
We defined a case as a resident of Halle with gastroenteritis, Cryptosporidium-positive stool and disease onset weeks 27 through 47. In a case-control study among kindergarten children, we compared cases and controls regarding environmental exposure, use of swimming pools, zoo visits and tap water consumption 14 days pre-onset or a corresponding 14-days-period (controls) and adjusted for residence. Stool specimens were tested by microscopy and PCR, and Cryptosporidium DNA was sequenced. Samples from public water system, swimming pools and river Saale were examined for Cryptosporidium oocysts (microscopy and PCR).
Overall, 167 cases were detected, 40/167 (24%) were classified as secondary cases. First disease onsets occurred during week 29, numbers peaked in week 34 and started to decrease in week 36. Median age was 8 years (range: 0-77). Compared to controls (n = 61), cases (n = 20) were more likely to report visits to previously flooded areas (OR: 4.9; 95%-CI: 1.4-18) and the zoo (OR: 2.6; 95%-CI: 0.9-7.6). In multivariable analysis visits to the floodplain remained the sole risk factor (OR: 5.5; 95%-CI: 1.4-22). Only C.hominis of a single genotype (IbA9G2) was detected in stools. Oocysts were detected in samples from the river, two local lakes and three public swimming pools by microscopy, but not in the public water supply.
Evidence suggests that activities in the dried out floodplain led to infection among children. Secondary transmissions may be involved. Consequently, authorities recommended to avoid playing, swimming and having picnics in the flood-affected area. Health authorities should consider the potential health risks of long-term surviving parasites persisting on flooded grounds and in open waters even several weeks after the flooding and of bathing places close to sewage spill-overs. Preventive measures comprise water sampling (involving parasites), information of the public and prolonged closures of potentially contaminated sites.
[Show abstract][Hide abstract] ABSTRACT: A pilot study was undertaken to investigate the occurrence of Cryptosporidium in four very small drinking water systems supplying communities in rural Puerto Rico. Water samples (40 L) were collected and oocysts were concentrated by calcium carbonate flocculation, recovered by immunomagnetic separation and detected by immunofluorescence microscopy. Cryptosporidium oocysts were identified in all four systems. This is the first report of evidence of the potential public health risk from this chlorine-resistant pathogen in Puerto Rican small water systems. Further work is warranted to fully assess the health risks that Cryptosporidium and other protozoa pose to populations served by community-managed small drinking water systems.
Journal of Water and Health 09/2015; 13(3):853-858. DOI:10.2166/wh.2015.223 · 1.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.
The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).
The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.
This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium, Eimeria and Toxoplasma species are single-celled, intracellular, spore forming parasites. They are understudied and whilst some species can be cultured in vitro, there is a lack of suitable culture systems for others such as Cryptosporidium spp. There is an urgent need for high-quality genomic sequences to aid in bio-marker discovery, in-silico drug-target/-vaccine screening, and geno-epidemiology.
Next generation sequencing (NGS) has the potential for increasing our knowledge of the biology, transmission, virulence and epidemiology of these species and so improve diagnosis, control and treatment of coccidial infections in animals and humans. However, significant challenges exist for generation of sequence data directly from animal and human samples. Parasite cells may be present in very low numbers, whilst samples are often contaminated with host/bacterial cells. Here we discuss the development of protocols to enrich for, and purify, Cryptosporidium oocysts from stool samples, optimisation of DNA extraction methods, the pro’s and con’s of whole genome amplification (WGA), and use of transposon based techniques to sequence sub-nano-gram quantities of genomic DNA. We have sequenced and created PAGIT-improved assemblies of several Cryptosporidium genomes directly from clinical samples, and characterised new human species i.e. C. viatorum - first isolated by Elwin et al, (2012), from patients with gastro-intestinal symptoms returning from the Indian sub-continent. We also discuss the application of these techniques to characterise Eimeria tenella and Toxoplasma gondii isolates.
Apicomplexa in farm animals International meeting, Edinburgh; 07/2015
[Show abstract][Hide abstract] ABSTRACT: We report a widespread foodborne outbreak of Cryptosporidium parvum in England and Scotland in May 2012. Cases were more common in female adults, and had no history of foreign travel. Over 300 excess cases were identified during the period of the outbreak. Speciation and microbiological typing revealed the outbreak strain to be C. parvum gp60 subtype IIaA15G2R1.
Hypothesis generation questionnaires were administered and an unmatched case control study was undertaken to test the hypotheses raised. Cases and controls were interviewed by telephone. Controls were selected using sequential digit dialling. Information was gathered on demographics, foods consumed and retailers where foods were purchased.
Seventy-four laboratory confirmed cases and 74 controls were included in analyses. Infection was found to be strongly associated with the consumption of pre-cut mixed salad leaves sold by a single retailer. This is the largest documented outbreak of cryptosporidiosis attributed to a food vehicle.
PLoS ONE 05/2015; 10(5):e0125955. DOI:10.1371/journal.pone.0125955 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the level of practice consistent with United Kingdom national standards for Cryptosporidium testing, an audit was performed of 156 publicly-funded clinical microbiology laboratories in England and Wales between August 2013 and April 2014. Responses were received from 85 (54%) laboratories. First line diagnostic methods used were mainly microscopy with modified Ziehl-Neelsen (mZN) or auramine phenol (AP) staining (68/85, 80%), enzyme immunoassays (EIAs) (16/85, 19%) or in-house PCR (1/85, 1%). The use of EIAs was more widespread than reported previously. Various methods were used for confirmation of positive EIA reactions and laboratories frequently resorted to sending samples to the national reference laboratory for this purpose, indicating that guidance is required for performance monitoring and confirmation of positive reactions. Laboratory positivity rates were related to the diagnostic test used, with highest median rates reported by those using PCR, EIAs or AP microscopy, and the lowest by those using mZN microscopy. One third of responding laboratories (28/85, 33%) routinely tested all stools for Cryptosporidium. However, 16 (19%) laboratories used stool consistency to decide whether to test for this parasite. Other selection criteria included patient age (n=18; 21% laboratories), history or clinical details (n=40; 47%), duration of hospitalisation (n=18; 21%) or clinician requests (n=25; 29%). To encourage laboratories to test all stools submitted for the investigation of diarrhoeal illness for Cryptosporidium, revision of the guidance in the national standards is underway. This will enable improved assessment of the burden of illness and ability to monitor outbreaks, and measure changes in reported cases.
Journal of Medical Microbiology 05/2015; 64(7). DOI:10.1099/jmm.0.000089 · 2.25 Impact Factor
[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.
[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). DOI:10.1007/s00436-014-3828-6 · 2.10 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. DOI:10.2166/wh.2013.097 · 1.46 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; 63(Pt_1). DOI:10.1099/jmm.0.066050-0 · 2.25 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; 62(Pt_10). DOI:10.1099/jmm.0.058743-0 · 2.25 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 (15/17 kDa and 27 kDa) using longitudinal sera taken from laboratory-confirmed cryptosporidiosis cases in England and Wales. The effect of the infecting Cryptosporidium species was also investigated.
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.
Overall responses to the 15/17 kDa antigen complex were stronger and over a greater range than those to the 27 kDa antigen, but declined between 181 and 240 days and were barely detectable thereafter. Responses to the 27 kDa 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.
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 15/17 kDa antigen occurring within 6 months of infection.
The Journal of infection 05/2013; 67(3). DOI:10.1016/j.jinf.2013.04.019 · 4.44 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; 29(5). DOI:10.1016/j.pt.2013.03.001 · 6.20 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.