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Unravelling Cryptosporidium and Giardia epidemiology
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Unravelling Cryptosporidium and Giardia epidemiology Simone M. Caccio`1, R.C. Andrew Thompson2, Jim McLauchlin3 and Huw V. Smith4 1
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena, 299, 00161 Rome, Italy 2 World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch 6150, Australia 3 Health Protection Agency, Food Safety Microbiology Laboratory, Centre for Infections, 61 Colindale Avenue, London, UK, NW9 5HT 4 Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, UK, G21 3UW
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.
suis and Cryptosporidium muris) and two genotypes (monkey and cervine) of Cryptosporidium are associated with human disease, and molecular approaches have enabled a greater understanding of the contributions of humans and livestock as reservoirs of infection. Using species-typing tools, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for C. parvum and C. hominis, the most commonly reported causes of human cryptosporidiosis. However, less progress has been made in elucidating the epidemiology of giardiasis; only Giardia duodenalis genotypes A and B have so far been associated with human infections. Both the role of animals in transmitting G. duodenalis to humans and the most likely routes of infection remain unclear.
Cryptosporidium and Giardia species as human pathogens Cryptosporidium and Giardia are genera of protozoan parasites that infect a wide range of vertebrates. Species within these genera cause human cryptosporidiosis and giardiasis, which probably constitute the most common causes of protozoal diarrhoea worldwide, and lead to significant morbidity and mortality in both the developing and developed world. Transmission is through the faecal– oral route following direct or indirect contact with the transmissive stages {Cryptosporidium oocysts and Giardia cysts [(oo)cysts]}, including person to person, zoonotic, waterborne, foodborne and airborne (for Cryptosporidium) transmission. Molecular biology has provided powerful new tools for characterizing Cryptosporidium and Giardia, and the analysis of previously unrecognized genetic differences within these genera has revolutionized both their taxonomy and our understanding of the epidemiology of human disease. Molecular tools have enabled not only the identification of species and genotypes in the faeces of infected hosts but also their recognition in environmental samples, including water. At least seven species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium
Taxonomy of Cryptosporidium and Giardia Early workers relied largely on host occurrence in describing species of Cryptosporidium and Giardia, which resulted in the description of a large number of species and a history of taxonomic confusion and controversy [1,2]. The lack of morphological characters to differentiate variants led to much debate over whether phenotypic differences were ‘real’ and reflected genetic differences, or were the result of environmental- or hostinduced changes [3]. The application of molecular tools has revealed that Giardia and Cryptosporidium are a phenotypically and genotypically heterogeneous assemblage of species and genotypes that are largely morphologically identical [4–6]. The genus Cryptosporidium now comprises 14 species (Table 1), along with several genotypes that show varying genetic diversity, in some cases greater than that observed between named species [5]. Giardia is similarly classified into six species, with further genetically distinct assemblages within G. duodenalis, which are likely to represent different species [6] (Table 1). The use of molecular approaches has had an enormous impact on elucidating the nature of variation in Giardia and Cryptosporidium, especially because they enable the direct characterization of cysts or oocysts recovered from faecal or environmental samples, thus avoiding bias due to preferential amplification by in vitro culture or animal
Corresponding author: Caccio`, S.M. ([email protected]). Available online 19 July 2005
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Table 1. Species of Cryptosporidium and Giardia Cryptosporidiuma Species C. hominis
Major host Humans, monkeys
C. parvum
Cattle, other ruminants, humans
C. andersoni
Cattle
C. muris
Rodents
C. suis
Pigs
C. felis
Cats
C. C. C. C. C. C. C. C.
Dogs Guinea pigs Poultry Turkeys, humans Finches, chicken Reptiles Lizard Fish
canis wrairi bailey meleagridis galli serpentis saurophilum molnari
Giardiab Speciesc G. duodenalisd Assemblage A G. duodenalis Assemblage B (G. enterica) G. duodenalis Assemblage C (G. canis) G. duodenalis Assemblage E (G. bovis) G. duodenalis Assemblage F (G. cati) G. duodenalis Assemblage G (G. simondi) G. agilis G. muris G. microti G. psittaci G. ardeae
Major host Humans, livestock Humans
Dogs
Cattle, other hoofed livestock
Cats
Rats
Amphibians Rodents Muskrats, voles Birds Birds
a
Details in Ref. [5]. Details in Ref. [6]. Other criteria used to distinguish Giardia species include morphology of the trophozoite by electron microscopy, host specificity and genetics. G. duodenalis assemblages have been proposed to be classified into six different species (see Ref. [6] for discussion). d G. duodenalis is a synonym for Giardia lamblia and Giardia intestinalis. b c
models. Species of Cryptosporidium and Giardia are now better defined, many with distinct host specificities. In addition, there is growing evidence for differences between species and genotypes within Giardia and Cryptosporidium with respect to parasite development, growth rates, drug sensitivity and other phenotypic characteristics, in addition to disease presentation. Many newly recognized species were previously described largely on the basis of host occurrence, and thus a nomenclature is already available, although as a result of the lack of ‘type’ (i.e. reference) material for comparison [5,6], a degree of supposition is required to make some of these associations. The taxonomy of both Cryptosporidium and Giardia remains only partially resolved, and the species status of genetic variants of both parasites (especially in the absence of other data) will be an important and controversial future issue to resolve. Trends in the epidemiology of cryptosporidiosis and giardiasis Common characteristics of both Cryptosporidium and Giardia markedly influence the epidemiology of these infections: (i) the infective dose is low [one to ten (oo)cysts] for both parasites; (ii) (oo)cysts are immediately infectious when excreted in faeces, and can be transmitted by person to person contact; (iii) (oo)cysts are remarkably stable and can survive for weeks to months in the environment; and (iv) environmental dispersal can lead to the contamination of drinking water and food. Direct and indirect transmission between infected hosts and susceptible individuals is favoured by high population densities (e.g. during lambing or calving), and by close contact (e.g. during www.sciencedirect.com
recreational bathing or consumption of contaminated water). Risk factors for sporadic cryptosporidiosis [7–10] include age (children under five years of age and, to a lesser extent, young adults, who presumably have a greater likelihood of contact with these patients), travelling abroad, contact with a diarrhoeic individual and contact with farm animals. Swimming in fresh water or public swimming pools were positively associated only in the Australian and US studies [7,8]. Hunter et al. [9] identified separate risks for C. hominis (travel abroad and contact with diarrhoeic individual) and C. parvum (contact with cattle). Joint and eye pain, recurrent headaches, dizzy spells and fatigue occurred significantly more often in C. hominis cases than in C. parvum cases [11]. Some evidence exists for larger numbers of C. hominis than C. parvum oocysts being excreted [12,13], which could prove important when estimating the impact of human sewage as a means by which oocysts are transmitted into the environment. Eating tomatoes and carrots was strongly negatively associated with infection in these studies, reinforcing similar data from outbreaks [10]. This negative association has not been explained adequately, although consumption of fruit and vegetables contaminated with small numbers of parasites might cause subclinical infection and, in turn, augment protective immunity. Similar studies have been conducted for sporadic giardiasis but in the absence of genotyping. In a UK case-control study [14], the main risk factors were swallowing water while swimming, drinking treated tap water, contact with recreational fresh water and eating
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Box 1. Genetic markers for studying Cryptosporidium and Giardia epidemiology † Genetic markers differ in their information content, and the nature of the DNA fragment selected for detecting and characterizing Cryptosporidium and Giardia should be considered carefully (Table 2). To detect species, analysis of highly or moderately conserved coding regions (e.g. 18S rDNA, structural and housekeeping genes) is required, whereas investigations into the transmission of genotypes and subtypes, identifying sources of infection and risk factors, requires more discriminatory fingerprinting techniques [such as those based on minisatellites (MN) and MS], which can identify individual isolates or clonal lineages. † The genome sequences of C. parvum [15], C. hominis [16] and G. duodenalis [17] are available, and it is now possible to ‘mine’ the genomic databases to identify sequences that are likely to show polymorphisms. Among these sequences, MN and MS are probably the best candidates. MN and MS DNA sequences consist of tandem repeats of a simple motif of variable length (10–100 base pairs for MN and 1–5 base pairs for MS) and are found throughout the genomes of all eukaryotes. MS exhibit length polymorphisms, resulting from either gain or loss of repeat units that occur during DNA replication. Consequently, alleles of different sizes are frequently observed at MS loci, enabling identification of unique genetic fingerprints. Recent work [18,19] has confirmed the use of MN and MS markers in the study of the population structure of Cryptosporidium and in understanding the transmission dynamics of infection [20]. † Typing and subtyping systems used for human and non-human samples should also be used for environmental samples, and
lettuce. Giardia cysts probably survive less well in raw water than do C. parvum oocysts, although cysts are usually detected more frequently and with higher abundance in raw and drinking water. The association with eating lettuce (and other fruit and vegetables which receive minimal heat treatment before consumption) also highlights the role of contaminated wastewater, uncomposted sewage sludge or manure used as fertiliser, in addition to direct contamination of produce by wildlife, or transmission vectors such as refuse-feeding birds and nonbiting filth flies. Molecular characterization of Cryptosporidium and Giardia The value of characterizing the genetic diversity of Cryptosporidium and Giardia at different levels of
particularly for source and disease tracking. This avoids the confusion of using different systems for human, non-human host and environmental samples for public health investigation of disease outbreaks. † Limitations in the number of procedures undertaken with environmental samples containing small numbers of oocysts and cysts, and hence small amounts of extractable DNA, will occur. For such samples, the most important information required by public health investigators will be species and assemblage identification. † Species and assemblage analysis should be undertaken on the basis of at least two loci because this provides the most robust information (R.A.B Nichols and H.V. Smith, unpublished). At least one locus should be 18S rDNA, and, where possible, another should be suitable for both species identification and further subtyping analysis. † For Cryptosporidium, MN and MS typing [18,19,21,22], gp60 sequencing [23,24] and analysis of a double-stranded RNA element [25] have been used to subtype C. parvum and C. hominis, and might offer sufficient subspecies discrimination to address public health investigations, either separately or in combination. † A standardized nomenclature is required to avoid confusion and to enable multicentre validation comparisons. This is less of an issue with gp60 sequencing than with satellite subtyping. † Although unavailable currently, similar discriminatory subtyping tools are urgently required for Giardia. † The most commonly used PCR assays for detecting and typing Cryptosporidium and Giardia are listed in Table 2.
specificity and the importance of appropriate nucleic acid analysis cannot be overemphasized. Molecular tools for inter- and intraspecies discrimination differ [15–25] (Box 1 and Table 2). Using interspecies discrimination tools to analyse more than 3000 stool samples (Table 3), C. hominis and C. parvum have been identified as the major causes of human cryptosporidiosis but their prevalence varies in different regions of the world. C. hominis is more prevalent (62%, on average) in North and South America, Australia and Africa, whereas C. parvum causes more human infections in Europe (57%, on average), especially in the UK [26–40] (Table 3). Geographical variation occurs also within countries [33], and molecular epidemiological studies indicate that the proportion of C. parvum infections in
Table 2. List of the targets, type of assay and main use of amplification-based techniques for Cryptosporidium and Giardiaa Amplification target Cryptosporidium 18S rDNA Hsp70 COWP Actin b-Tubulin GP60 Microsatellites Minisatellites Extrachromosomal double-stranded RNA Giardia 16S rDNA GDH TPI b-Giardin EF-1a GLORF-C4
Assay type
Main application
PCR, nested PCR, sequencing, PCR-RFLP, real-time PCR, microarray PCR, nested PCR, sequencing, real-time PCR, microarray PCR, nested PCR, sequencing, PCR-RFLP, microarray PCR, nested PCR, sequencing PCR, nested PCR, sequencing, PCR-RFLP PCR, nested PCR, sequencing PCR, nested PCR, sequencing, fragment typing PCR, nested PCR, sequencing, fragment typing Reverse transcriptase, PCR, sequencing, heteroduplex mobility assays
Species and genotype identification Species and genotype identification Species and genotype identification Species and genotype identification Species and genotype identification Subgenotype identification Subgenotype identification Subgenotype identification Subgenotype identification
PCR, PCR, PCR, PCR, PCR, PCR,
Species and genotype identification Species and genotype identification Species and genotype identification Genotype identification Species and genotype identification Genotype identification
nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing sequencing, PCR-RFLP
microarray PCR-RFLP real-time PCR, microarray PCR-RFLP, real-time PCR, microarray
a Abbreviations: COWP, Cryptosporidium oocyst wall protein; EF-1a, elongation factor 1 a; GDH, glutamate dehydrogenase; GLORF-C4, G. lamblia open reading frame C4; GP60, glycoprotein 60; Hsp70, heat shock protein 70; RFLP, restriction fragment length polymorphism; TPI, triose phosphate isomerase.
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Table 3. Occurrence of Cryptosporidium species in immunocompetent and immunocompromised individuals Cryptosporidium species in immunocompetent individuals Country Cases C. parvum C. hominis (numbers) Czech Republic Children (9) 9 2 Czech Republic nka (3) Denmark Adult (44) 18 25 France Northern Ireland Switzerland Switzerland The Netherlands
Adult (11) Adult (39) Children (14) nk (9) Adult (29)
7 35 3 9 7 b
England Scotland
Various (2414 ) Various (137)
1354 64
Canada Japan Japan Japan Nepal Kenya Malawi Malawi
nk (150) nk (1) nk (3) All (22) nk (2) All (9) Children (9) Children (43)
29 1 2 3
Peru Brazil New Zealand
Children (118) Children (7) nk (423)
20
Total
3496
8 1
Italy France France Switzerland Portugal England Total
0 1
1
C. canis
Cervine genotype
Monkey genotype
22 b
1005 71
22
6
1 2
108 1 16 2 9 6 41
9
3
10
223
76 7 198
1798 (51%)
1606 (46%)
36 (1%)
Cryptosporidium species in immunocompromised individuals Country Cases C. parvum C. hominis (numbers) Vietnam (3) 3 Thailand (29) 24 Thailand (34) 5 17 Peru (299) 34 204 Kenya (6) 1 4 (24) (2) (4) (17) (10) (21)
C. felis
4 4 11
3 2
Kenya Malawi Guatemala USA USA South Africa
C. meleagridis
4
9 d
2 11 (0.3%)
C. meleagridis
C. felis
3 7 38 1
1 3 10
1
2 1 5
14 1 4 15 5 16
(9) (46) (13) (13)
8 22 7 7
14 6 2
3
1 6
1
3
(40) (27) 597
22 17 140 (23%)
11 1 341 (57%)
3 7 64 (11%)
4 2 33 (6%)
12 (0.3%)
C. canis
9 (0.2%)
C. muris
C. suis
1
1
3
1
1
15 (2%)
3
Refs
18s rDNA, COWP 18s rDNA, hsp70 18s rDNA, COWP, MS 18s rDNA TRAP-C2 18s rDNA Hsp70 b-Tubulin, COWP, Gp900 COWP, 18S rDNA 18s rDNA, COWP, MS 18s rDNA, COWP 18s rDNA GP60 18s rDNA, Gp900 GP60 18s rDNA 18s rDNA 18s rDNA, hsp70, GP60 18s rDNA, hsp70 18s rDNA b-Tubulin, 18S rDNA
[26] [27] [22] [28] [29] [30] [31] [32] [33] andc [18] [34] [35] [36] [37] [36] [38] [38] [39] [13] [38] [40]
2
1 2 12
PCR target
PCR target
Refs
18s rDNA 18s rDNA 18s rDNA 18s rDNA 18s rDNA, hsp70, acetyl-CoA 18s rDNA 18s rDNA TRAP-C2 TRAP-C2 18s rDNA TRAP, COWP, GP60 COWP, MS 18s rDNA Rep DNA 18s rDNA, hsp70, acetyl-CoA 18s rDNA COWP, 18S rDNA
[38] [41] [42] [43] [44] [38] [38] [45] [45] [46] [24] [21,47] [28] [48] [44] [49] c
1
a
Not known. b Additional 21 cases with both C. hominis and C. parvum. c J. McLauchlin et al., unpublished. d Genotypes closely related to C. canis.
humans is much higher in rural than in urban areas [40]. Although the reasons for this difference are not known, intensive livestock husbandry, coupled with communal birthing and feeding of large numbers of susceptible calves and lambs, can, at least partially, explain the spread of livestock-derived C. parvum into humans [5]. In the UK, a marked bimodal seasonal pattern of disease has been described: one peak during the spring and the second during late summer to early autumn [33]. The spring peak is almost exclusively due to C. parvum, whereas both C. parvum and C. hominis occur in the late summer to early autumn peak. Finding C. parvum in humans does not provide conclusive evidence for zoonotic transmission. Subgenotyping analysis (Box 1) of human C. parvum isolates identified genetic variants that are rarely found in other animals, suggesting that many C. parvum infections in www.sciencedirect.com
particular areas might have originated from humans themselves [5]. Host substructuring was also demonstrated in an analysis of Scottish human and bovine C. parvum isolates using microsatellite (MS) typing [18,19], whereby C. parvum subgroups 1 and 5 seemed to be ‘human specific’, suggesting the existence of different C. parvum populations. Further micro- and macroepidemiological analyses are required to determine whether host substructuring is commonplace. C. meleagridis, C. canis, C. felis, C. suis, C. muris and the cervine and monkey genotypes of Cryptosporidium also infect immunocompetent and immunocompromised humans [6,18,21,24,28,34,36,41–49] (Table 3). In particular, C. meleagridis, a parasite originally described in turkeys [5], is now recognized as an emerging human pathogen, being responsible for 1% of all infections in the UK (Table 3) and w10% in Peru, where its prevalence is as
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high as for C. parvum [43]. The implication of these results is that farm animals, domestic pets and some wildlife are potential sources of infection for humans, particularly (but not exclusively) for immunocompromised individuals. The quantity and quality of current genotyping tools limits the understanding of the molecular epidemiology of human giardiasis (Table 2), and the direct characterization of human and non-human-derived cysts have mostly been based on analysis of single genetic loci [50–62]. Based on a clonal population structure, some authors consider the use of a single, highly polymorphic marker suitable for epidemiological investigations, comparable to multilocus sequence typing in terms of resolution [54]. However, clonality in G. duodenalis has not been supported by convincing statistical analysis, mainly as a result of an insufficient number of samples tested [63]. The single genetic locus approach therefore remains to be validated. Samples from different geographical locations, examined by PCR amplification of DNA extracted from faeces (Table 4), confirm that only G. duodenalis assemblages A and B are associated with human infections. The prevalence of each assemblage varies considerably from country to country; assemblage B being more common overall (Table 4), but no strong conclusions can be drawn from current data. Giardiasis is associated with a spectrum of symptomatology, including acute and chronic malabsorptive and allergic manifestations and childhood failure to thrive, but whether these are host, parasite or host–parasite influenced remains unknown. The exact contribution of parasite genetic variability to symptomatology is still under debate because of contrasting results linking assemblage A to mild, intermittent diarrhoea and assemblage B to severe, acute or persistent diarrhoea [52], or vice versa [56]. This is an important area for further investigation. Zoonotic transmission Transmission of Cryptosporidium and Giardia can be direct, from host to host, or indirect, through the
ingestion of contaminated food or water. A multitude of transmission cycles therefore exist, involving domestic animals and wildlife, which in some instances result in human infections. Understanding how these cycles interact and the frequency of transmission requires molecular epidemiological studies in defined endemic locations. Early reports of human cryptosporidiosis drew attention to zoonotic transmission (contact with infected young cattle or sheep and consumption of livestockcontaminated drinking water) [64,65], and its importance was stressed recently by the observation of a dramatic decline (81%) in the reported incidence of human C. parvum cryptosporidiosis in the UK, coinciding with a foot and mouth disease outbreak. During the spring of 2001, strict restrictions on access to the countryside were imposed, which reduced human contact with livestock, feral animals and their faeces [66]. Because touching farm animals is a risk factor associated with C. parvum infection [9], the observed decline could be explained by the removal of access to the countryside, and, as a result of livestock movement restriction and culling, a reduced input of oocysts into water catchment areas occurred [67]. A concurrent decrease in giardiasis was not detected during this period, suggesting that this parasite has significantly different reservoirs of infection and/or routes of transmission, at least in the UK [67]. Although cattle have been repeatedly implicated as sources of waterborne cryptosporidiosis outbreaks, genotyping the contaminating isolate(s) has often implicated human effluent as the source, as with the notorious Milwaukee outbreak [20]. However, livestock has been implicated as the source of waterborne outbreaks: cattle in the Cranbrook (Canada) outbreak [68] and sheep in three outbreaks in England [33]. In a Scottish study of sporadic human cryptosporidiosis, C. parvum was the aetiological agent in 84% of 67 cases, supporting livestock faecal pollution of water sources as the leading cause of sporadic cryptosporidiosis [69]. Direct contact with animals [33,68]
Table 4. Occurrence of G. duodenalis assemblages A and B in human faecal samples
a
Country Italy Wales England and Wales The Netherlands Ohio, USA California, USA Canada Australia
Sample type (numbers) Sporadic (30) Nursery outbreak (21) Sporadic (1185) Population survey (18) Sporadic (14) Sporadic (2) Waterborne outbreak (6) Sporadic (8)
Assemblage A 24 (80%)
Australia Australia China Korea Laos India India
Population survey (23 children) Sporadic (12) Sporadic (8) Sporadic (5) Sporadic (5) Sporadic (10) Sporadic (19)
7 (30%)
6 (32%)
Peru Uganda Turkey Total
Sporadic (35) Sporadic (3) Sporadic (44) 1438
6 (24%) 3 (100%) 19 (43%) 370 (26%)
J. McLauchlin et al., unpublished.
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265 (22%) 9 (50%) 14 (100%)
Assemblage B 6 (20%) 21 (100%) 843 (71%) 9 (50%)
Mixed ACB
77 (7%)
2 (100%) 6 (100%) 2 (25%)
4 (50%) 5 (100%)
6 (75%) 16 (70%) 11 (92%) 4 (50%) 5 (100%) 10 (100%) 9 (47%)
1 (8%)
4 (21%)
19 (76%) 25 (57%) 986 (69%)
82 (6%)
PCR target b-giardin TPI TPI GDH 16S rDNA TPI 16S rDNA 16S rDNA GDH 16S rDNA 16S rDNA 16S rDNA 16S rDNA GLORF-C4 TPI TPI EF1-a TPI 16S rDNA TPI
Refs [50] [51] a
[52] [53] [54] [53] [55] [56] [57] [58] [58] [59] [54] [60] [54] [61] [62]
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or contaminated food [70] has also been linked to outbreaks of C. parvum cryptosporidiosis in humans in North America and Europe. Companion animals have long been considered as potential sources for human cryptosporidiosis and giardiasis. Despite the frequency with which infected patients have household pets, pets have rarely been implicated as the source of infection. Dogs and cats seem to be most commonly infected with the predominantly host-adapted C. canis and C. felis [35], and, as such, dogs, cats and other companion animals might not be important zoonotic reservoirs of Cryptosporidium infection. Further molecular characterization of oocysts from infected animals in endemic areas is required before this assumption can be verified. The zoonotic potential of the various G. duodenalis assemblages has been controversial for many years. Genotyping data suggest that the public health risk from cattle is probably minimal, at least in North America and Australia, where the livestock genotype (G. duodenalis assemblage E or Giardia bovis; Table 1) predominates [54,71,72]. Cattle are susceptible to transient infection with zoonotic genotypes in which the frequency of transmission with the livestock genotype is high and competition is likely to occur. Domestic dogs are susceptible to infection with both host-adapted (G. duodenalis assemblage C or G. canis; Table 1) and zoonotic Giardia genotypes, and recent molecular epidemiological studies demonstrate zoonotic transmission in an endemic focus in situations where humans and dogs live closely together [60]. However, the epidemiological circumstances that support zoonotic transmission remain to be resolved and this requires further molecular epidemiological studies in well defined endemic locations. The occurrence of Giardia in wildlife, particularly of isolates that are morphologically identical to G. duodenalis, has been the single most important factor implicating Giardia as a zoonotic agent. It is therefore surprising that there is little evidence to support the role of wildlife as a source of disease in humans. Although wildlife, particularly aquatic mammals, is commonly infected with Giardia, there is little evidence to implicate such infections as the original contaminating source in waterborne outbreaks [4]. It would seem that such animals are more likely to have become infected from water contaminated with faecal material of human or, less likely, domestic animal origin, thus serving to amplify the numbers of the originally contaminating isolate. The few studies that have genotyped Giardia of beaver origin have confirmed previous suggestions that the source of Giardia infection in beavers was likely to be of human origin [54,73]. Future perspectives Molecular tools provide new insights into Cryptosporidium and Giardia taxonomy and have helped to unravel their complex epidemiologies. At least seven Cryptosporidium species and two Cryptosporidium genotypes, in addition to two G. duodenalis genotypes, cause human disease. The uncertainty of their zoonotic potential, particularly for G. duodenalis, complicates the issue, given the numerous transmission routes and the low www.sciencedirect.com
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infectious doses for humans. Identifying environmental sources of contamination and routes of infection, particularly for human giardiasis, requires an ‘in depth’ understanding of which genotypes are host adapted (and do not infect humans) and which are transmissible to humans. Characterization of Giardia DNA of animal origin is urgently required, particularly aquatic animals, given the abundance of waterborne cysts and the scarcity of waterborne outbreaks. Molecular assays must be applicable to clinical (human and non-human hosts) and environmental (including food) samples, particularly for species and genotype identity and source, and disease tracking. Specific requirements for developing effective molecular identification and genotyping systems in environmental samples include increased discrimination, specificity and sensitivity, which can be exploited to improve understanding of the epidemiology of infection, disease and outbreak investigations.
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in raw wastewater in Milwaukee: implications for understanding outbreak occurrence and transmission dynamics. J. Clin. Microbiol. 41, 5254–5257 Caccio`, S. et al. (2000) A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology 120, 237–244 Enemark, H.L. et al. (2002) Molecular characterization of Danish Cryptosporidium parvum isolates. Parasitology 125, 331–341 Strong, W.B. et al. (2000) Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infect. Immun. 68, 4117–4134 Leav, B.A. et al. (2002) Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among Cryptosporidium isolates from human immunodeficiency virus-infected children in South Africa. Infect. Immun. 70, 3881–3890 Leoni, F. et al. (2003) Molecular epidemiological analysis of Cryptosporidium isolates from humans and animals by using a heteroduplex mobility assay and nucleic acid sequencing based on a small double-stranded RNA element. J. Clin. Microbiol. 41, 981–992 Hajdusek, O. et al. (2004) Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic. Vet. Parasitol. 122, 183–192 Ryan, U. et al. (2003) Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl. Environ. Microbiol. 69, 4302–4307 Guyot, K. et al. (2001) Molecular characterization of Cryptosporidium isolates obtained from humans in France. J. Clin. Microbiol. 39, 3472–3480 Lowery, C.J. et al. (2001) Molecular genotyping of human cryptosporidiosis in Northern Ireland: epidemiological aspects and review. Ir. J. Med. Sci. 170, 246–250 Glaeser, C. et al. (2004) Detection and molecular characterization of Cryptosporidium spp. isolated from diarrheic children in Switzerland. Pediatr. Infect. Dis. J. 23, 359–361 Fretz, R. et al. (2003) Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland. Epidemiol. Infect. 131, 663–667 Caccio`, S. et al. (1999) Genetic polymorphism at the b-tubulin locus among human and animal isolates of Cryptosporidium parvum. FEMS Microbiol. Lett. 170, 173–179 McLauchlin, J. et al. (2000) Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J. Clin. Microbiol. 38, 3984–3990 Ong, C.S. et al. (2002) Novel cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype. Emerg. Infect. Dis. 8, 263–268 Abe, N. et al. (2002) Cryptosporidium infection in dogs in Osaka, Japan. Vet. Parasitol. 108, 185–193 Wu, Z. et al. (2003) Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses. Appl. Environ. Microbiol. 69, 4720–4726 Yagita, K. et al. (2001) Molecular characterization of Cryptosporidium isolates obtained from human and bovine infections in Japan. Parasitol. Res. 87, 950–955 Gatei, W. et al. (2003) Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. J. Clin. Microbiol. 41, 1458–1462 Peng, M.M. et al. (2003) Molecular epidemiology of cryptosporidiosis in children in Malawi. J. Eukaryot. Microbiol. 50(Suppl.), 557–559 Learmonth, J.J. et al. (2004) Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand. Appl. Environ. Microbiol. 70, 3973–3978 Tiangtip, R. and Jongwutiwes, S. (2002) Molecular analysis of Cryptosporidium species isolated from HIV-infected patients in Thailand. Trop. Med. Int. Health 7, 357–364 Gatei, W. et al. (2002) Zoonotic species of Cryptosporidium are as prevalent as the anthroponotic in HIV-infected patients in Thailand. Ann. Trop. Med. Parasitol. 96, 797–802
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43 Cama, V.A. et al. (2003) Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru. J. Eukaryot. Microbiol. 50(Suppl.), 531–533 44 Morgan, U. et al. (2000) Molecular characterization of Cryptosporidium isolates obtained from human immunodeficiency virusinfected individuals living in Switzerland, Kenya, and the United States. J. Clin. Microbiol. 38, 1180–1183 45 Sulaiman, I.M. et al. (1998) Differentiating human from animal isolates of Cryptosporidium parvum. Emerg. Infect. Dis. 4, 681–685 46 Pieniazek, N.J. et al. (1999) New Cryptosporidium genotypes in HIV-infected persons. Emerg. Infect. Dis. 5, 444–449 47 Caccio`, S. et al. (2002) Human infection with Cryptosporidium felis: case report and literature review. Emerg. Infect. Dis. 8, 85–86 48 Bonnin, A. et al. (1996) Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence. FEMS Microbiol. Lett. 137, 207–211 49 Matos, O. et al. (2004) Cryptosporidium felis and C. meleagridis in persons with HIV, Portugal. Emerg. Infect. Dis. 10, 2256–2257 50 Caccio`, S.M. et al. (2002) Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int. J. Parasitol. 32, 1023–1030 51 Amar, C.F. et al. (2002) Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. J. Clin. Microbiol. 40, 446–452 52 Homan, W.L. and Mank, T.G. (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31, 822–826 53 Van Keulen, H. et al. (2002) Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis. Vet. Parasitol. 108, 97–107 54 Sulaiman, I.M. et al. (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg. Infect. Dis. 9, 1444–1452 55 Read, C.M. et al. (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect. Genet. Evol. 4, 125–130 56 Read, C. et al. (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. Int. J. Parasitol. 32, 229–231 57 Hopkins, R.M. et al. (1997) Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J. Parasitol. 83, 44–51 58 Yong, T.S. et al. (2000) Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA sequences. J. Parasitol. 86, 887–891 59 Yong, T.S. et al. (2002) PCR-RFLP analysis of Giardia intestinalis using a Giardia-specific gene, GLORF-C4. Parasite 9, 65–70 60 Traub, R.J. et al. (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128, 253–262 61 Graczyk, T.K. et al. (2002) Anthropozoonotic Giardia duodenalis genotype (Assemblage A) infections in habitats of free-ranging human-habituated gorillas, Uganda. J. Parasitol. 88, 905–909 62 Aydin, A.F. et al. (2004) Classification of Giardia duodenalis parasites in Turkey into groups A and B using restriction fragment length polymorphism. Diagn. Microbiol. Infect. Dis. 50, 147–151 63 Monis, P.T. et al. (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect. Genet. Evol. 3, 29–38 64 Current, W.L. et al. (1983) Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission. N. Engl. J. Med. 308, 1252–1257 65 Department of the Environment, Department of Health (1995) Cryptosporidium in Water Supplies. Second Report of the Group of Experts; Chairman, Sir John Badenoch, HMSO 66 Hunter, P.R. et al. (2003) Foot and mouth disease and cryptosporidiosis: possible interaction between two emerging infectious diseases. Emerg. Infect. Dis. 9, 109–112 67 Smerdon, W.J. et al. (2003) Foot and mouth disease in livestock and reduced cryptosporidiosis in humans, England and Wales. Emerg. Infect. Dis. 9, 22–28
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68 Fayer, R. et al. (2000) Epidemiology of Cryptosporidium: transmission, detection and identification. Int. J. Parasitol. 30, 1305–1322 69 Goh, S. et al. (2004) Sporadic cryptosporidiosis, North Cumbria, England, 1996–2000. Emerg. Infect. Dis. 10, 1007–1015 70 Stantic-Pavlinic, M. et al. (2003) Cryptosporidiosis associated with animal contacts. Wien. Klin. Wochenschr. 115, 125–127 71 O’Handley, R.M. et al. (2000) Prevalence and genotypic characterisation of Giardia in dairy calves from Western Australia and Western Canada. Vet. Parasitol. 90, 193–200
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72 Hoar, B.R. et al. (2001) An examination of risk factors associated with beef cattle shedding pathogens of potential zoonotic concern. Epidemiol. Infect. 127, 147–155 73 Appelbee, A. et al. (2002) Genotypic characterization of Giardia cysts isolated from wild beaver in southern Alberta, Canada. In Giardia: The Cosmopolitan Parasite (Olson, B.E. et al., eds), pp. 299–300, CAB International
Elsevier celebrates two anniversaries with gift to university libraries in the developing world In 1580, the Elzevir family began their printing and bookselling business in the Netherlands, publishing works by scholars such as John Locke, Galileo Galilei and Hugo Grotius. On 4 March 1880, Jacobus George Robbers founded the modern Elsevier company intending, just like the original Elzevir family, to reproduce fine editions of literary classics for the edification of others who shared his passion, other ’Elzevirians’. Robbers co-opted the Elzevir family’s old printer’s mark, visually stamping the new Elsevier products with a classic old symbol of the symbiotic relationship between publisher and scholar. Elsevier has since become a leader in the dissemination of scientific, technical and medical (STM) information, building a reputation for excellence in publishing, new product innovation and commitment to its STM communities. In celebration of the House of Elzevir’s 425th anniversary and the 125th anniversary of the modern Elsevier company, Elsevier will donate books to 10 university libraries in the developing world. Entitled ‘A Book in Your Name’, each of the 6 700 Elsevier employees worldwide has been invited to select one of the chosen libraries to receive a book donated by Elsevier. The core gift collection contains the company’s most important and widely used STM publications including Gray’s Anatomy, Dorland’s Illustrated Medical Dictionary, Essential Medical Physiology, Cecil Essentials of Medicine, Mosby’s Medical, Nursing and Allied Health Dictionary, The Vaccine Book, Fundamentals of Neuroscience, and Myles Textbook for Midwives. The 10 beneficiary libraries are located in Africa, South America and Asia. They include the Library of the Sciences of the University of Sierra Leone; the library of the Muhimbili University College of Health Sciences of the University of Dar es Salaam, Tanzania; the library of the College of Medicine of the University of Malawi; and the libraries of the University of Zambia, Universite du Mali, Universidade Eduardo Mondlane, Mozambique; Makerere University, Uganda; Universidad San Francisco de Quito, Ecuador; Universidad Francisco Marroquin, Guatemala; and the National Centre for Scientific and Technological Information (NACESTI), Vietnam. Through ‘A Book in Your Name’, the 10 libraries will receive approximately 700 books at a retail value of approximately 1 million US dollars.
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Unravelling Cryptosporidium and Giardia epidemiology Simone M. Caccio`1, R.C. Andrew Thompson2, Jim McLauchlin3 and Huw V. Smith4 1
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena, 299, 00161 Rome, Italy 2 World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch 6150, Australia 3 Health Protection Agency, Food Safety Microbiology Laboratory, Centre for Infections, 61 Colindale Avenue, London, UK, NW9 5HT 4 Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, UK, G21 3UW
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.
suis and Cryptosporidium muris) and two genotypes (monkey and cervine) of Cryptosporidium are associated with human disease, and molecular approaches have enabled a greater understanding of the contributions of humans and livestock as reservoirs of infection. Using species-typing tools, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for C. parvum and C. hominis, the most commonly reported causes of human cryptosporidiosis. However, less progress has been made in elucidating the epidemiology of giardiasis; only Giardia duodenalis genotypes A and B have so far been associated with human infections. Both the role of animals in transmitting G. duodenalis to humans and the most likely routes of infection remain unclear.
Cryptosporidium and Giardia species as human pathogens Cryptosporidium and Giardia are genera of protozoan parasites that infect a wide range of vertebrates. Species within these genera cause human cryptosporidiosis and giardiasis, which probably constitute the most common causes of protozoal diarrhoea worldwide, and lead to significant morbidity and mortality in both the developing and developed world. Transmission is through the faecal– oral route following direct or indirect contact with the transmissive stages {Cryptosporidium oocysts and Giardia cysts [(oo)cysts]}, including person to person, zoonotic, waterborne, foodborne and airborne (for Cryptosporidium) transmission. Molecular biology has provided powerful new tools for characterizing Cryptosporidium and Giardia, and the analysis of previously unrecognized genetic differences within these genera has revolutionized both their taxonomy and our understanding of the epidemiology of human disease. Molecular tools have enabled not only the identification of species and genotypes in the faeces of infected hosts but also their recognition in environmental samples, including water. At least seven species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium
Taxonomy of Cryptosporidium and Giardia Early workers relied largely on host occurrence in describing species of Cryptosporidium and Giardia, which resulted in the description of a large number of species and a history of taxonomic confusion and controversy [1,2]. The lack of morphological characters to differentiate variants led to much debate over whether phenotypic differences were ‘real’ and reflected genetic differences, or were the result of environmental- or hostinduced changes [3]. The application of molecular tools has revealed that Giardia and Cryptosporidium are a phenotypically and genotypically heterogeneous assemblage of species and genotypes that are largely morphologically identical [4–6]. The genus Cryptosporidium now comprises 14 species (Table 1), along with several genotypes that show varying genetic diversity, in some cases greater than that observed between named species [5]. Giardia is similarly classified into six species, with further genetically distinct assemblages within G. duodenalis, which are likely to represent different species [6] (Table 1). The use of molecular approaches has had an enormous impact on elucidating the nature of variation in Giardia and Cryptosporidium, especially because they enable the direct characterization of cysts or oocysts recovered from faecal or environmental samples, thus avoiding bias due to preferential amplification by in vitro culture or animal
Corresponding author: Caccio`, S.M. ([email protected]). Available online 19 July 2005
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Table 1. Species of Cryptosporidium and Giardia Cryptosporidiuma Species C. hominis
Major host Humans, monkeys
C. parvum
Cattle, other ruminants, humans
C. andersoni
Cattle
C. muris
Rodents
C. suis
Pigs
C. felis
Cats
C. C. C. C. C. C. C. C.
Dogs Guinea pigs Poultry Turkeys, humans Finches, chicken Reptiles Lizard Fish
canis wrairi bailey meleagridis galli serpentis saurophilum molnari
Giardiab Speciesc G. duodenalisd Assemblage A G. duodenalis Assemblage B (G. enterica) G. duodenalis Assemblage C (G. canis) G. duodenalis Assemblage E (G. bovis) G. duodenalis Assemblage F (G. cati) G. duodenalis Assemblage G (G. simondi) G. agilis G. muris G. microti G. psittaci G. ardeae
Major host Humans, livestock Humans
Dogs
Cattle, other hoofed livestock
Cats
Rats
Amphibians Rodents Muskrats, voles Birds Birds
a
Details in Ref. [5]. Details in Ref. [6]. Other criteria used to distinguish Giardia species include morphology of the trophozoite by electron microscopy, host specificity and genetics. G. duodenalis assemblages have been proposed to be classified into six different species (see Ref. [6] for discussion). d G. duodenalis is a synonym for Giardia lamblia and Giardia intestinalis. b c
models. Species of Cryptosporidium and Giardia are now better defined, many with distinct host specificities. In addition, there is growing evidence for differences between species and genotypes within Giardia and Cryptosporidium with respect to parasite development, growth rates, drug sensitivity and other phenotypic characteristics, in addition to disease presentation. Many newly recognized species were previously described largely on the basis of host occurrence, and thus a nomenclature is already available, although as a result of the lack of ‘type’ (i.e. reference) material for comparison [5,6], a degree of supposition is required to make some of these associations. The taxonomy of both Cryptosporidium and Giardia remains only partially resolved, and the species status of genetic variants of both parasites (especially in the absence of other data) will be an important and controversial future issue to resolve. Trends in the epidemiology of cryptosporidiosis and giardiasis Common characteristics of both Cryptosporidium and Giardia markedly influence the epidemiology of these infections: (i) the infective dose is low [one to ten (oo)cysts] for both parasites; (ii) (oo)cysts are immediately infectious when excreted in faeces, and can be transmitted by person to person contact; (iii) (oo)cysts are remarkably stable and can survive for weeks to months in the environment; and (iv) environmental dispersal can lead to the contamination of drinking water and food. Direct and indirect transmission between infected hosts and susceptible individuals is favoured by high population densities (e.g. during lambing or calving), and by close contact (e.g. during www.sciencedirect.com
recreational bathing or consumption of contaminated water). Risk factors for sporadic cryptosporidiosis [7–10] include age (children under five years of age and, to a lesser extent, young adults, who presumably have a greater likelihood of contact with these patients), travelling abroad, contact with a diarrhoeic individual and contact with farm animals. Swimming in fresh water or public swimming pools were positively associated only in the Australian and US studies [7,8]. Hunter et al. [9] identified separate risks for C. hominis (travel abroad and contact with diarrhoeic individual) and C. parvum (contact with cattle). Joint and eye pain, recurrent headaches, dizzy spells and fatigue occurred significantly more often in C. hominis cases than in C. parvum cases [11]. Some evidence exists for larger numbers of C. hominis than C. parvum oocysts being excreted [12,13], which could prove important when estimating the impact of human sewage as a means by which oocysts are transmitted into the environment. Eating tomatoes and carrots was strongly negatively associated with infection in these studies, reinforcing similar data from outbreaks [10]. This negative association has not been explained adequately, although consumption of fruit and vegetables contaminated with small numbers of parasites might cause subclinical infection and, in turn, augment protective immunity. Similar studies have been conducted for sporadic giardiasis but in the absence of genotyping. In a UK case-control study [14], the main risk factors were swallowing water while swimming, drinking treated tap water, contact with recreational fresh water and eating
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Box 1. Genetic markers for studying Cryptosporidium and Giardia epidemiology † Genetic markers differ in their information content, and the nature of the DNA fragment selected for detecting and characterizing Cryptosporidium and Giardia should be considered carefully (Table 2). To detect species, analysis of highly or moderately conserved coding regions (e.g. 18S rDNA, structural and housekeeping genes) is required, whereas investigations into the transmission of genotypes and subtypes, identifying sources of infection and risk factors, requires more discriminatory fingerprinting techniques [such as those based on minisatellites (MN) and MS], which can identify individual isolates or clonal lineages. † The genome sequences of C. parvum [15], C. hominis [16] and G. duodenalis [17] are available, and it is now possible to ‘mine’ the genomic databases to identify sequences that are likely to show polymorphisms. Among these sequences, MN and MS are probably the best candidates. MN and MS DNA sequences consist of tandem repeats of a simple motif of variable length (10–100 base pairs for MN and 1–5 base pairs for MS) and are found throughout the genomes of all eukaryotes. MS exhibit length polymorphisms, resulting from either gain or loss of repeat units that occur during DNA replication. Consequently, alleles of different sizes are frequently observed at MS loci, enabling identification of unique genetic fingerprints. Recent work [18,19] has confirmed the use of MN and MS markers in the study of the population structure of Cryptosporidium and in understanding the transmission dynamics of infection [20]. † Typing and subtyping systems used for human and non-human samples should also be used for environmental samples, and
lettuce. Giardia cysts probably survive less well in raw water than do C. parvum oocysts, although cysts are usually detected more frequently and with higher abundance in raw and drinking water. The association with eating lettuce (and other fruit and vegetables which receive minimal heat treatment before consumption) also highlights the role of contaminated wastewater, uncomposted sewage sludge or manure used as fertiliser, in addition to direct contamination of produce by wildlife, or transmission vectors such as refuse-feeding birds and nonbiting filth flies. Molecular characterization of Cryptosporidium and Giardia The value of characterizing the genetic diversity of Cryptosporidium and Giardia at different levels of
particularly for source and disease tracking. This avoids the confusion of using different systems for human, non-human host and environmental samples for public health investigation of disease outbreaks. † Limitations in the number of procedures undertaken with environmental samples containing small numbers of oocysts and cysts, and hence small amounts of extractable DNA, will occur. For such samples, the most important information required by public health investigators will be species and assemblage identification. † Species and assemblage analysis should be undertaken on the basis of at least two loci because this provides the most robust information (R.A.B Nichols and H.V. Smith, unpublished). At least one locus should be 18S rDNA, and, where possible, another should be suitable for both species identification and further subtyping analysis. † For Cryptosporidium, MN and MS typing [18,19,21,22], gp60 sequencing [23,24] and analysis of a double-stranded RNA element [25] have been used to subtype C. parvum and C. hominis, and might offer sufficient subspecies discrimination to address public health investigations, either separately or in combination. † A standardized nomenclature is required to avoid confusion and to enable multicentre validation comparisons. This is less of an issue with gp60 sequencing than with satellite subtyping. † Although unavailable currently, similar discriminatory subtyping tools are urgently required for Giardia. † The most commonly used PCR assays for detecting and typing Cryptosporidium and Giardia are listed in Table 2.
specificity and the importance of appropriate nucleic acid analysis cannot be overemphasized. Molecular tools for inter- and intraspecies discrimination differ [15–25] (Box 1 and Table 2). Using interspecies discrimination tools to analyse more than 3000 stool samples (Table 3), C. hominis and C. parvum have been identified as the major causes of human cryptosporidiosis but their prevalence varies in different regions of the world. C. hominis is more prevalent (62%, on average) in North and South America, Australia and Africa, whereas C. parvum causes more human infections in Europe (57%, on average), especially in the UK [26–40] (Table 3). Geographical variation occurs also within countries [33], and molecular epidemiological studies indicate that the proportion of C. parvum infections in
Table 2. List of the targets, type of assay and main use of amplification-based techniques for Cryptosporidium and Giardiaa Amplification target Cryptosporidium 18S rDNA Hsp70 COWP Actin b-Tubulin GP60 Microsatellites Minisatellites Extrachromosomal double-stranded RNA Giardia 16S rDNA GDH TPI b-Giardin EF-1a GLORF-C4
Assay type
Main application
PCR, nested PCR, sequencing, PCR-RFLP, real-time PCR, microarray PCR, nested PCR, sequencing, real-time PCR, microarray PCR, nested PCR, sequencing, PCR-RFLP, microarray PCR, nested PCR, sequencing PCR, nested PCR, sequencing, PCR-RFLP PCR, nested PCR, sequencing PCR, nested PCR, sequencing, fragment typing PCR, nested PCR, sequencing, fragment typing Reverse transcriptase, PCR, sequencing, heteroduplex mobility assays
Species and genotype identification Species and genotype identification Species and genotype identification Species and genotype identification Species and genotype identification Subgenotype identification Subgenotype identification Subgenotype identification Subgenotype identification
PCR, PCR, PCR, PCR, PCR, PCR,
Species and genotype identification Species and genotype identification Species and genotype identification Genotype identification Species and genotype identification Genotype identification
nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing, nested PCR, sequencing sequencing, PCR-RFLP
microarray PCR-RFLP real-time PCR, microarray PCR-RFLP, real-time PCR, microarray
a Abbreviations: COWP, Cryptosporidium oocyst wall protein; EF-1a, elongation factor 1 a; GDH, glutamate dehydrogenase; GLORF-C4, G. lamblia open reading frame C4; GP60, glycoprotein 60; Hsp70, heat shock protein 70; RFLP, restriction fragment length polymorphism; TPI, triose phosphate isomerase.
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Table 3. Occurrence of Cryptosporidium species in immunocompetent and immunocompromised individuals Cryptosporidium species in immunocompetent individuals Country Cases C. parvum C. hominis (numbers) Czech Republic Children (9) 9 2 Czech Republic nka (3) Denmark Adult (44) 18 25 France Northern Ireland Switzerland Switzerland The Netherlands
Adult (11) Adult (39) Children (14) nk (9) Adult (29)
7 35 3 9 7 b
England Scotland
Various (2414 ) Various (137)
1354 64
Canada Japan Japan Japan Nepal Kenya Malawi Malawi
nk (150) nk (1) nk (3) All (22) nk (2) All (9) Children (9) Children (43)
29 1 2 3
Peru Brazil New Zealand
Children (118) Children (7) nk (423)
20
Total
3496
8 1
Italy France France Switzerland Portugal England Total
0 1
1
C. canis
Cervine genotype
Monkey genotype
22 b
1005 71
22
6
1 2
108 1 16 2 9 6 41
9
3
10
223
76 7 198
1798 (51%)
1606 (46%)
36 (1%)
Cryptosporidium species in immunocompromised individuals Country Cases C. parvum C. hominis (numbers) Vietnam (3) 3 Thailand (29) 24 Thailand (34) 5 17 Peru (299) 34 204 Kenya (6) 1 4 (24) (2) (4) (17) (10) (21)
C. felis
4 4 11
3 2
Kenya Malawi Guatemala USA USA South Africa
C. meleagridis
4
9 d
2 11 (0.3%)
C. meleagridis
C. felis
3 7 38 1
1 3 10
1
2 1 5
14 1 4 15 5 16
(9) (46) (13) (13)
8 22 7 7
14 6 2
3
1 6
1
3
(40) (27) 597
22 17 140 (23%)
11 1 341 (57%)
3 7 64 (11%)
4 2 33 (6%)
12 (0.3%)
C. canis
9 (0.2%)
C. muris
C. suis
1
1
3
1
1
15 (2%)
3
Refs
18s rDNA, COWP 18s rDNA, hsp70 18s rDNA, COWP, MS 18s rDNA TRAP-C2 18s rDNA Hsp70 b-Tubulin, COWP, Gp900 COWP, 18S rDNA 18s rDNA, COWP, MS 18s rDNA, COWP 18s rDNA GP60 18s rDNA, Gp900 GP60 18s rDNA 18s rDNA 18s rDNA, hsp70, GP60 18s rDNA, hsp70 18s rDNA b-Tubulin, 18S rDNA
[26] [27] [22] [28] [29] [30] [31] [32] [33] andc [18] [34] [35] [36] [37] [36] [38] [38] [39] [13] [38] [40]
2
1 2 12
PCR target
PCR target
Refs
18s rDNA 18s rDNA 18s rDNA 18s rDNA 18s rDNA, hsp70, acetyl-CoA 18s rDNA 18s rDNA TRAP-C2 TRAP-C2 18s rDNA TRAP, COWP, GP60 COWP, MS 18s rDNA Rep DNA 18s rDNA, hsp70, acetyl-CoA 18s rDNA COWP, 18S rDNA
[38] [41] [42] [43] [44] [38] [38] [45] [45] [46] [24] [21,47] [28] [48] [44] [49] c
1
a
Not known. b Additional 21 cases with both C. hominis and C. parvum. c J. McLauchlin et al., unpublished. d Genotypes closely related to C. canis.
humans is much higher in rural than in urban areas [40]. Although the reasons for this difference are not known, intensive livestock husbandry, coupled with communal birthing and feeding of large numbers of susceptible calves and lambs, can, at least partially, explain the spread of livestock-derived C. parvum into humans [5]. In the UK, a marked bimodal seasonal pattern of disease has been described: one peak during the spring and the second during late summer to early autumn [33]. The spring peak is almost exclusively due to C. parvum, whereas both C. parvum and C. hominis occur in the late summer to early autumn peak. Finding C. parvum in humans does not provide conclusive evidence for zoonotic transmission. Subgenotyping analysis (Box 1) of human C. parvum isolates identified genetic variants that are rarely found in other animals, suggesting that many C. parvum infections in www.sciencedirect.com
particular areas might have originated from humans themselves [5]. Host substructuring was also demonstrated in an analysis of Scottish human and bovine C. parvum isolates using microsatellite (MS) typing [18,19], whereby C. parvum subgroups 1 and 5 seemed to be ‘human specific’, suggesting the existence of different C. parvum populations. Further micro- and macroepidemiological analyses are required to determine whether host substructuring is commonplace. C. meleagridis, C. canis, C. felis, C. suis, C. muris and the cervine and monkey genotypes of Cryptosporidium also infect immunocompetent and immunocompromised humans [6,18,21,24,28,34,36,41–49] (Table 3). In particular, C. meleagridis, a parasite originally described in turkeys [5], is now recognized as an emerging human pathogen, being responsible for 1% of all infections in the UK (Table 3) and w10% in Peru, where its prevalence is as
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high as for C. parvum [43]. The implication of these results is that farm animals, domestic pets and some wildlife are potential sources of infection for humans, particularly (but not exclusively) for immunocompromised individuals. The quantity and quality of current genotyping tools limits the understanding of the molecular epidemiology of human giardiasis (Table 2), and the direct characterization of human and non-human-derived cysts have mostly been based on analysis of single genetic loci [50–62]. Based on a clonal population structure, some authors consider the use of a single, highly polymorphic marker suitable for epidemiological investigations, comparable to multilocus sequence typing in terms of resolution [54]. However, clonality in G. duodenalis has not been supported by convincing statistical analysis, mainly as a result of an insufficient number of samples tested [63]. The single genetic locus approach therefore remains to be validated. Samples from different geographical locations, examined by PCR amplification of DNA extracted from faeces (Table 4), confirm that only G. duodenalis assemblages A and B are associated with human infections. The prevalence of each assemblage varies considerably from country to country; assemblage B being more common overall (Table 4), but no strong conclusions can be drawn from current data. Giardiasis is associated with a spectrum of symptomatology, including acute and chronic malabsorptive and allergic manifestations and childhood failure to thrive, but whether these are host, parasite or host–parasite influenced remains unknown. The exact contribution of parasite genetic variability to symptomatology is still under debate because of contrasting results linking assemblage A to mild, intermittent diarrhoea and assemblage B to severe, acute or persistent diarrhoea [52], or vice versa [56]. This is an important area for further investigation. Zoonotic transmission Transmission of Cryptosporidium and Giardia can be direct, from host to host, or indirect, through the
ingestion of contaminated food or water. A multitude of transmission cycles therefore exist, involving domestic animals and wildlife, which in some instances result in human infections. Understanding how these cycles interact and the frequency of transmission requires molecular epidemiological studies in defined endemic locations. Early reports of human cryptosporidiosis drew attention to zoonotic transmission (contact with infected young cattle or sheep and consumption of livestockcontaminated drinking water) [64,65], and its importance was stressed recently by the observation of a dramatic decline (81%) in the reported incidence of human C. parvum cryptosporidiosis in the UK, coinciding with a foot and mouth disease outbreak. During the spring of 2001, strict restrictions on access to the countryside were imposed, which reduced human contact with livestock, feral animals and their faeces [66]. Because touching farm animals is a risk factor associated with C. parvum infection [9], the observed decline could be explained by the removal of access to the countryside, and, as a result of livestock movement restriction and culling, a reduced input of oocysts into water catchment areas occurred [67]. A concurrent decrease in giardiasis was not detected during this period, suggesting that this parasite has significantly different reservoirs of infection and/or routes of transmission, at least in the UK [67]. Although cattle have been repeatedly implicated as sources of waterborne cryptosporidiosis outbreaks, genotyping the contaminating isolate(s) has often implicated human effluent as the source, as with the notorious Milwaukee outbreak [20]. However, livestock has been implicated as the source of waterborne outbreaks: cattle in the Cranbrook (Canada) outbreak [68] and sheep in three outbreaks in England [33]. In a Scottish study of sporadic human cryptosporidiosis, C. parvum was the aetiological agent in 84% of 67 cases, supporting livestock faecal pollution of water sources as the leading cause of sporadic cryptosporidiosis [69]. Direct contact with animals [33,68]
Table 4. Occurrence of G. duodenalis assemblages A and B in human faecal samples
a
Country Italy Wales England and Wales The Netherlands Ohio, USA California, USA Canada Australia
Sample type (numbers) Sporadic (30) Nursery outbreak (21) Sporadic (1185) Population survey (18) Sporadic (14) Sporadic (2) Waterborne outbreak (6) Sporadic (8)
Assemblage A 24 (80%)
Australia Australia China Korea Laos India India
Population survey (23 children) Sporadic (12) Sporadic (8) Sporadic (5) Sporadic (5) Sporadic (10) Sporadic (19)
7 (30%)
6 (32%)
Peru Uganda Turkey Total
Sporadic (35) Sporadic (3) Sporadic (44) 1438
6 (24%) 3 (100%) 19 (43%) 370 (26%)
J. McLauchlin et al., unpublished.
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265 (22%) 9 (50%) 14 (100%)
Assemblage B 6 (20%) 21 (100%) 843 (71%) 9 (50%)
Mixed ACB
77 (7%)
2 (100%) 6 (100%) 2 (25%)
4 (50%) 5 (100%)
6 (75%) 16 (70%) 11 (92%) 4 (50%) 5 (100%) 10 (100%) 9 (47%)
1 (8%)
4 (21%)
19 (76%) 25 (57%) 986 (69%)
82 (6%)
PCR target b-giardin TPI TPI GDH 16S rDNA TPI 16S rDNA 16S rDNA GDH 16S rDNA 16S rDNA 16S rDNA 16S rDNA GLORF-C4 TPI TPI EF1-a TPI 16S rDNA TPI
Refs [50] [51] a
[52] [53] [54] [53] [55] [56] [57] [58] [58] [59] [54] [60] [54] [61] [62]
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or contaminated food [70] has also been linked to outbreaks of C. parvum cryptosporidiosis in humans in North America and Europe. Companion animals have long been considered as potential sources for human cryptosporidiosis and giardiasis. Despite the frequency with which infected patients have household pets, pets have rarely been implicated as the source of infection. Dogs and cats seem to be most commonly infected with the predominantly host-adapted C. canis and C. felis [35], and, as such, dogs, cats and other companion animals might not be important zoonotic reservoirs of Cryptosporidium infection. Further molecular characterization of oocysts from infected animals in endemic areas is required before this assumption can be verified. The zoonotic potential of the various G. duodenalis assemblages has been controversial for many years. Genotyping data suggest that the public health risk from cattle is probably minimal, at least in North America and Australia, where the livestock genotype (G. duodenalis assemblage E or Giardia bovis; Table 1) predominates [54,71,72]. Cattle are susceptible to transient infection with zoonotic genotypes in which the frequency of transmission with the livestock genotype is high and competition is likely to occur. Domestic dogs are susceptible to infection with both host-adapted (G. duodenalis assemblage C or G. canis; Table 1) and zoonotic Giardia genotypes, and recent molecular epidemiological studies demonstrate zoonotic transmission in an endemic focus in situations where humans and dogs live closely together [60]. However, the epidemiological circumstances that support zoonotic transmission remain to be resolved and this requires further molecular epidemiological studies in well defined endemic locations. The occurrence of Giardia in wildlife, particularly of isolates that are morphologically identical to G. duodenalis, has been the single most important factor implicating Giardia as a zoonotic agent. It is therefore surprising that there is little evidence to support the role of wildlife as a source of disease in humans. Although wildlife, particularly aquatic mammals, is commonly infected with Giardia, there is little evidence to implicate such infections as the original contaminating source in waterborne outbreaks [4]. It would seem that such animals are more likely to have become infected from water contaminated with faecal material of human or, less likely, domestic animal origin, thus serving to amplify the numbers of the originally contaminating isolate. The few studies that have genotyped Giardia of beaver origin have confirmed previous suggestions that the source of Giardia infection in beavers was likely to be of human origin [54,73]. Future perspectives Molecular tools provide new insights into Cryptosporidium and Giardia taxonomy and have helped to unravel their complex epidemiologies. At least seven Cryptosporidium species and two Cryptosporidium genotypes, in addition to two G. duodenalis genotypes, cause human disease. The uncertainty of their zoonotic potential, particularly for G. duodenalis, complicates the issue, given the numerous transmission routes and the low www.sciencedirect.com
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infectious doses for humans. Identifying environmental sources of contamination and routes of infection, particularly for human giardiasis, requires an ‘in depth’ understanding of which genotypes are host adapted (and do not infect humans) and which are transmissible to humans. Characterization of Giardia DNA of animal origin is urgently required, particularly aquatic animals, given the abundance of waterborne cysts and the scarcity of waterborne outbreaks. Molecular assays must be applicable to clinical (human and non-human hosts) and environmental (including food) samples, particularly for species and genotype identity and source, and disease tracking. Specific requirements for developing effective molecular identification and genotyping systems in environmental samples include increased discrimination, specificity and sensitivity, which can be exploited to improve understanding of the epidemiology of infection, disease and outbreak investigations.
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72 Hoar, B.R. et al. (2001) An examination of risk factors associated with beef cattle shedding pathogens of potential zoonotic concern. Epidemiol. Infect. 127, 147–155 73 Appelbee, A. et al. (2002) Genotypic characterization of Giardia cysts isolated from wild beaver in southern Alberta, Canada. In Giardia: The Cosmopolitan Parasite (Olson, B.E. et al., eds), pp. 299–300, CAB International
Elsevier celebrates two anniversaries with gift to university libraries in the developing world In 1580, the Elzevir family began their printing and bookselling business in the Netherlands, publishing works by scholars such as John Locke, Galileo Galilei and Hugo Grotius. On 4 March 1880, Jacobus George Robbers founded the modern Elsevier company intending, just like the original Elzevir family, to reproduce fine editions of literary classics for the edification of others who shared his passion, other ’Elzevirians’. Robbers co-opted the Elzevir family’s old printer’s mark, visually stamping the new Elsevier products with a classic old symbol of the symbiotic relationship between publisher and scholar. Elsevier has since become a leader in the dissemination of scientific, technical and medical (STM) information, building a reputation for excellence in publishing, new product innovation and commitment to its STM communities. In celebration of the House of Elzevir’s 425th anniversary and the 125th anniversary of the modern Elsevier company, Elsevier will donate books to 10 university libraries in the developing world. Entitled ‘A Book in Your Name’, each of the 6 700 Elsevier employees worldwide has been invited to select one of the chosen libraries to receive a book donated by Elsevier. The core gift collection contains the company’s most important and widely used STM publications including Gray’s Anatomy, Dorland’s Illustrated Medical Dictionary, Essential Medical Physiology, Cecil Essentials of Medicine, Mosby’s Medical, Nursing and Allied Health Dictionary, The Vaccine Book, Fundamentals of Neuroscience, and Myles Textbook for Midwives. The 10 beneficiary libraries are located in Africa, South America and Asia. They include the Library of the Sciences of the University of Sierra Leone; the library of the Muhimbili University College of Health Sciences of the University of Dar es Salaam, Tanzania; the library of the College of Medicine of the University of Malawi; and the libraries of the University of Zambia, Universite du Mali, Universidade Eduardo Mondlane, Mozambique; Makerere University, Uganda; Universidad San Francisco de Quito, Ecuador; Universidad Francisco Marroquin, Guatemala; and the National Centre for Scientific and Technological Information (NACESTI), Vietnam. Through ‘A Book in Your Name’, the 10 libraries will receive approximately 700 books at a retail value of approximately 1 million US dollars.
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