Crypto Di Eropa.pdf

REVIEW

Human cryptosporidiosis in Europe S. M. Cacciò1 and R. M. Chalmers2 1) European Union Reference Laboratory for Parasites, Istituto Superiore di Sanità, Rome, Italy and 2) Cryptosporidium Reference Unit, Public Health Wales, Singleton Hospital, Swansea, UK

Abstract Cryptosporidium has emerged as a significant cause of diarrhoeal disease worldwide, with severe health consequences for very young, malnourished children living in endemic areas and for individuals with highly impaired T-cell functions. In Europe, as elsewhere, the burden of disease has been difficult to measure as a result of the lack of appropriate, standardized surveillance and monitoring systems. The recent occurrence of large water- and foodborne outbreaks in several EU countries, as well as the results of many surveys of human and animal cryptosporidiosis, indicate that this parasite is widespread. Specific subtypes of the zoonotic Cryptosporidium parvum and the anthroponotic C. hominis are responsible for the majority of human cases in Europe. No treatment is currently available to clear the infection, but recent progress in genetic engineering of the parasite, coupled with advances in genomics, have opened important avenues for future research. Here we explore the possible reasons for underascertainment of cryptosporidiosis and the importance of accurate diagnosis in clinical management, the epidemiology of human cryptosporidiosis and key messages from recent outbreaks to highlight important interventions and emerging public health issues. © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Keywords: Cryptosporidiosis, epidemiology, food, outbreaks, water Article published online: 10 May 2016

Corresponding author: S. M. Cacciò, European Union Reference Laboratory for Parasites, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy E-mail: [email protected]

Introduction The protozoan parasite Cryptosporidium (Phylum Apicomplexa) is a well-established cause of sporadic gastroenteritis, as well as outbreaks characterized by watery diarrhoea, abdominal pain, nausea, vomiting and low-grade fever. The parasite has a global distribution, and a number of species are recognized as human pathogens, although most cases are due to Cryptosporidium hominis and C. parvum [1]. For the profoundly immunocompromised, such as very young infants, people who are severely malnourished or people who have a coexisting health problem (e.g. untreated HIV infection) which leads to T-cell immunodeficiency, symptoms can be severe, prolonged and even lifethreatening [2]. Furthermore, infection with Cryptosporidium

can cause persistent symptoms in immunocompetent subjects that extend beyond the acute illness, as shown by case–control studies in the United Kingdom [3] and Sweden [4]. Some of these symptoms may be indicative of postinfectious irritable bowel syndrome. Two studies have reported that experimental C. parvum infection of rats triggered long-term pathologic changes in the lining of the small intestine very similar to those found in human patients with irritable bowel syndrome [5,6]. Reactive arthropathy has also been reported after cryptosporidiosis [7,8], and in one study in the United Kingdom it was more likely to be reported as due to C. hominis than C. parvum [3]. Nevertheless, Cryptosporidium is underdiagnosed and underreported in most countries, despite being one of the communicable diseases for which surveillance is mandatory in the European Union (EU) and European Economic Area (EEA) countries. This is due to several factors, including healthcareseeking behaviour by patients, access to relevant services, poor awareness in the primary care setting of its role as a cause of gastrointestinal symptoms leading to a low request rate for specific testing, variable provision of diagnostic tests and reporting practices and the lack of harmonized, EU-wide,

Clin Microbiol Infect 2016; 22: 471–480 © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved http://dx.doi.org/10.1016/j.cmi.2016.04.021

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surveillance or monitoring programs (Fig. 1). Individual EU and EEA countries upload data from their own surveillance systems to the European Surveillance System (TESSy) for publication in the annual epidemiologic report for food- and waterborne diseases and zoonoses published by the European Centre for Disease Control (ECDC). The most recently published 2014 report, which contains data for 2012 [9], showed that only 19 of 27 EU countries reported cryptosporidiosis case, whereas cryptosporidiosis is not subjected to official notification in Denmark, France, Greece, Italy, the Netherlands and Portugal, and Belgium and Spain do not have a system that covers the whole population [9]. Five countries reported zero cases, two reported just one case, and only seven reported 50 or more cases [9].

Diagnosis and Clinical Management Although cryptosporidiosis is unpleasant and debilitating, and lasts longer (more than 10 days) than episodes of

gastroenteritis caused by many viruses and bacteria (several days), in immunocompetent people, it is self-limiting. Accurate diagnosis is helpful for clinicians, patients and their carers so that they understand that symptoms of cryptosporidiosis may persist for a prolonged period and may relapse and remit during this time [3]. The high numbers of infective oocysts shed in faeces and the low infectious dose mean that Cryptosporidium is highly infectious, requiring specific measures for the prevention of person-to-person spread. For example, UK guidance [10] states that cases should not attend day care centres, food handlers and carers of highly susceptible patients should be excluded from work until 48 hours after diarrhoea has stopped, and patients should avoid using swimming pools for 2 weeks after the first normal stool because shedding of oocysts can continue after cessation of diarrhoea. Secondary transmission is worthy of further investigation, and influencing factors such as age, comorbidity and infectivity potential of specific subtypes need to be considered. Personal hygiene is regarded as a key intervention [11].

Reported Cryptosporidium cases and estimation of ascertainment parameters and multipliers in the EU countries [13] Germany

Factors influencing ascertainment parameters for Cryptosporidium, with examples

Stool analysed for Cryptosporidium

314 (1.93)

4128 (8.25)

• SensiƟvity of laboratory methods, assumed to be 50% for microscopy [13]

0.99

1.00

0.69

• NaƟonal guidance on tesƟng • SelecƟon criteria for tesƟng

0.18

0.11

1.00

0.34

0.10

0.34

0.08

0.16

35 000 (43)

54 178 (330)

62 698 (130)

109 000 (130) 100

651 201 (4000) 2100

385 961 (770) 93

Case submits a stool sample

Case visits a GP

• Cost to paƟent • Cost to GP • Ease of submission • symptom severity, duraƟon • opƟons for selfmanagement • age and sex . cost

All symptomaƟc cases of gastroenteriƟs (at least three loose stools, or any vomiƟng, in 24 h; excluding diagnosed non-infecƟous causes) a

b

England

1084 (1.29)

Cases reported Diagnosis reported to surveillance

The Netherlands

Mean annual reported cases in 2001-2005 (rate)a

Median probability of parameter occurring

0.19

Estimated median annual Crypto cases (rate)a Multiplierb

rate per 100 000 populaƟon the mulƟplier converts each reported case to the esƟmated number of symptomaƟc cases in the populaƟon.

FIG. 1. Surveillance pyramid illustrates where and how underdiagnosis and underreporting of Cryptosporidium in community diarrhoea cases affects reported data from European Union member states. Reported data underestimate true incidence, which vary by country [12]. Multipliers vary by country, but this is due in part to uncertainties in parameters used to reconstruct surveillance pyramid. Factors that contribute most to underreporting and underdiagnosis of Cryptosporidium are differences in healthcare use and laboratory practice. © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 471–480

Cacciò and Chalmers

Genuine differences in the occurrence of cryptosporidiosis may be reflected in case rates but health systems, diagnostic practice and reporting varies within and between countries, influencing surveillance data, and direct comparisons between countries may be misleading [12] as illustrated in Fig. 1; it is therefore important to understand where the variations lie. The ECDC’s 2014 annual epidemiologic report [9] considered that a lack of laboratory diagnosis was an important element of underreporting cryptosporidiosis cases, which adversely influences the detection of outbreaks and provision of advice for prevention of spread. It is important to recognize that Cryptosporidium oocysts are too small to be detected reliably during examination of faecal samples for ova, cysts and parasites. Specific tests are available, including acid-fast or fluorescent stains to stain faecal smears, enzyme immunoassays, immunochromatographic lateral flow assays and molecular methods [13]. However, not all laboratories test for Cryptosporidium in the diagnosis of diarrhoea, and many will only test certain patient groups (e.g. young children, HIV patients and travellers returning from endemic countries) or if specifically asked to do so [14]. Diagnostic methods have their limitations. Firstly, they may not be sufficiently sensitive for diagnosing and monitoring the most vulnerable patients who may require treatment, especially methods based on tinctorial stains or rapid lateral flow immunochromatographic assays [13]. Secondly, they do not distinguish between species. Only molecular tests identify species and genotypes, and are undertaken in some specialist and reference laboratories [13]. In addition to increased laboratory testing, speciation and subtyping of Cryptosporidium isolates were considered important for improved understanding of the epidemiology of cryptosporidiosis in the EU/EEA [9].

Epidemiology of Cryptosporidiosis The epidemiology of human cryptosporidiosis is complex, involving direct (person-to-person and animal-to-person) and indirect (through water, food and fomites contaminated with infectious oocysts) transmission routes (Fig. 2). Drinking untreated water, consuming water during recreational water activities, toileting young children or changing diapers, touching another person with diarrhoea and engaging in contact with farm animals increase the risk of becoming infected with Cryptosporidium spp. [1,15]. The main risk factors for C. hominis are linked to contact with young children, people with diarrhoea or contamination of water by human waste or wastewater. Although C. parvum can also be spread between people, the main risk factors are linked to contact with farm animals, especially young stock (e.g. at petting farms), or consumption of water or food contaminated by their faeces. Several recent

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FIG. 2. Graphic representation of main elements involved in epidemiology of Cryptosporidium hominis (left) and Cryptosporidium parvum (right) in Europe.

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reduction in the number of young farm animals and restrictions of the movement of both farm animals and people in the countryside. However, the sustained reduction in the spring peak was later found to be largely due to improved drinking water supplies [19]. On the other hand, the late summer–early autumn peak is mainly due to C. hominis and has not reduced in recent years; it is likely linked to increased travel and exposure to recreational water at this time of year [16]. Further discrimination of isolates is needed to improve our understanding of the epidemiology and transmission of Cryptosporidium. A literature survey showed that the results of genotyping by sequencing the highly polymorphic gp60 gene of 928 C. parvum and 1043 C. hominis isolates from humans in Europe has been published (Table 1) [15]. Variability in the sequence of this marker has been useful for inferring transmission routes; in addition, different C. hominis and C. parvum gp60 subtypes have been linked to variable clinical outcomes, producing different spectra in children [27] and in HIV-positive adults [28]. Notably, a single gp60 allelic family largely predominates among C. parvum isolates (family IIa, 84.8%) and another among C. hominis isolates (family Ib, 90.8%). Exceptions include patients with gastrointestinal disorders, where a greater variety of C. hominis gp60 families have been reported, and Sweden where the C. parvum and C. hominis appear to be more diverse than in other countries studied so far in Europe (Table 1). Further genetic variability exists even within allelic families, allowing the identification of gp60 subtypes, for which a specific

reviews provide more information on the epidemiology of cryptosporidiosis [1,15]. The ECDC’s annual epidemiologic reports contain yearly trends, age and gender distributions, and seasonality for reporting EU and EEA countries [9]. Worldwide, cryptosporidiosis is mostly a paediatric infection, and this is true for Europe as well. The highest prevalence among children under 5 years likely reflects increased exposure to the parasite as a result of poorer hygiene as well as a lack of, or only partial, immunity. However, it is important to note that infected children are likely to transmit the parasite to their parents. Indeed, a second, smaller, peak in prevalence is observed among adults aged 30 to 40 years old, especially women, and C. hominis is the predominant species involved [16]. As with young children, the elderly may be more susceptible to infection and vulnerable to its consequences (e.g. prolonged diarrhoea can upset electrolyte balance) [17]. Data from the ECDC, and especially the United Kingdom, indicate that cryptosporidiosis exhibits a strong seasonality in Europe, with low endemic levels followed by pronounced seasonal increases, particularly during late spring and late summer–early autumn. In the United Kingdom, springtime cases are more often due to C. parvum and are likely the result of an increased exposure to oocysts shed by young animals, as this coincides with the calving and lambing seasons [18]. In recent years, the spring peak has decreased in the United Kingdom. This was initially explained by the control measures for an outbreak of foot-and-mouth disease in 2001, involving

TABLE 1. Distribution of Cryptosporidium parvum and Cryptosporidium hominis gp60 families in humans in Europe C. parvum cases subtyped EU country

Patient group

N

IIa

IIb

Belgium France Ireland Ireland Italy Italy Netherlands Northern Ireland Portugal Portugal Romania Slovak Republic Slovenia Slovenia Slovenia Spain Spain Spain Spain Sweden Sweden Switzerland UK UK UK UK UK UK

Sporadic cases Waterborne outbreak Sporadic cases Sporadic cases Unspecified patients AIDS patients Sporadic cases Waterborne outbreak, sporadic cases Unspecified HIV-positive adults Children Family members Children and adults Patients with gastrointestinal disorders Unspecified Children with gastrointestinal disorders Patients with gastrointestinal disorders Sporadic cases Patients with gastrointestinal disorders 2 foodborne outbreaks Sporadic cases HIV positive Sporadic and outbreaks Unspecified Sporadic cases Outbreak cases Patients from farms Sporadic cases

6 4 0 1 1 0 79 78 0 170 170 0 None identified 8 4 0 13 9 0 52 52 0 4 1 0 25 9 1 4 0 0 Not investigated 31 29 0 21 21 0 3 3 0 3 3 0 164 146 0 7 6 0 12 7 0 27 4 0 107 69 0 2 1 0 87 82 0 None identified 69 56 0 22 22 0 11 11 0 Not investigated

C. hominis subtyped

IIc

IId

IIe

other

N

Ia

Ib

1 0 0 0

1 0 1 0

0 0 0 0

0 0 0 0

4 1 0 3 7

0 3 0 0 8 4

0 0 0 0 0 0

0 0 0 0 0 0

1 0 0 0 0 0 1 0 11 0 1

0 0 0 0 3 1 4 23 24 1 1

0 0 0 0 0 0 0 0 1 0 0

1 0 0 0 IIn (14) 0 0 0 IIo (2) 0 IIg (3)

1 0 0

9 0 0

0 0 0

3 0 0

13 0 13 22 0 21 25 0 25 Not investigated 5 0 5 1 1 0 64 0 63 68 1 67 3 2 0 15 1 10 Not investigated 1 0 1 2 1 1 71 0 53 None identified 41 6 35 318 3 289 60 0 54 89 0 85 2 0 1 63 5 44 3 0 2 65 16 46 3 0 3 Not investigated Not investigated 101 5 93

Updated from reference [15].

© 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 22, 471–480

0

Id

Ie

If

Ig

Reference

0 1 0

0 0 0

0 0 0

0 0 0

0 0 1 0 0 1

0 0 0 0 0 2

0 0 0 0 1 1

0 0 0 0 0 0

0 0 12

0 0 0

0 0 1

0 0 5

0 23 1 0 1 10 1 2 0

0 3 1 4 0 1 0 1 0

0 0 2 0 0 3 0 0 0

0 0 0 0 0 0 0 0 0

0

1

2

[15] [15] [15] [15] [15] [15] [15] [15] [15] [15] [20] [15] [15] [15] [15] [21] [22] [23] [24] [15] [15] [15] [25,26] [15] [15] [15] [15] [15]

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nomenclature has been proposed [29]. Direct zoonotic transmission of C. parvum is well documented in Europe, and several IIa subtypes—including IIaA15G2R1, which is particularly prevalent in young cattle; IIaA19G1R1, which has caused outbreaks where lambs were present [11,30]; and IIaA20G2R1 [31]—have been implicated in both sporadic cases and outbreaks involving animals (Table 2). Notably, subtypes belonging to allelic family IId also cause human cryptosporidiosis (Tables 1 and 2). This family is also often responsible for infection of lambs but has been reported in cattle in Sweden. In contrast, the C. hominis subtype IbA10G2 largely predominates in sporadic and outbreak cases in Europe (Fig. 2, Tables 1 and 2). This subtype is highly prevalent worldwide and has caused outbreaks in Australia and the United States, and thus it may be endowed with a particular virulence or a higher transmissibility compared to other subtypes [44]. While gp60 is certainly a useful epidemiologic marker, the discrimination among parasite isolates is increased by the use of a multilocus subtyping scheme, particularly when highly polymorphic markers (mini- and microsatellites) are included [45]. However, there is still no standardized multilocus subtyping scheme for Cryptosporidium. The establishment of such a scheme would be valuable for both interlaboratory surveillance and outbreak investigations.

Outbreaks of Cryptosporidiosis in Europe The identification of outbreaks of cryptosporidiosis is reliant on accurate diagnosis and surveillance, which was established formally in some countries much earlier than in others. For example, in England and Wales, surveillance for Cryptosporidium cases began in 1983 and for outbreaks of infectious intestinal disease in 1992 [46]. Over the last decade, outbreaks of cryptosporidiosis have been detected in many EU countries associated with drinking and recreational waters (mainly swimming pools), food consumption, animal contact, outdoor activities and person-to-person spread in the home and in institutions [47–49]. However, Cryptosporidium is often considered and tested only after bacterial and viral causes of symptoms have been excluded [35]. Here we have used data from selected, recent outbreaks to highlight some emerging aspects of cryptosporidiosis (Table 2). Firstly, waterborne outbreaks of cryptosporidiosis can be very large, and this has allowed analytical epidemiologic studies to investigate the long-term health sequelae, which are probably underestimated. In 2010–2011, Sweden registered the two largest outbreaks ever reported in Europe, together affecting an estimated 47 000 persons [39,40]. Both outbreaks were caused by C. hominis gp60 subtype IbA10G2. High consumption of tap

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water or living in a specific water supply region were identified as the most important risk factors for having cryptosporidiosis, and the high attack rate (28–45%) confirmed the high infectivity of this C. hominis subtype and the susceptibility of the local population to infection and disease. An important follow-up study revealed the extent of postinfection health consequences 2.5 to 8.5 months after the outbreak in Östersund, and 2.5 to 11.5 months after the outbreak in Skellefteå [39]. Outbreak cases were more likely to occur in patients reporting diarrhoea, watery diarrhoea, abdominal pain, joint pain, fatigue and nausea compared to nonoutbreak cases, and indicate a significant burden of illness even after outbreaks are over. Some differences were observed between the two sites: cases in Östersund also reported weight loss, loss of appetite, stiff joints and headache and generally stronger measures of association than in Skellefteå, which the authors speculatively attributed to the possibly greater contamination of the water supply leading to more frequent and severe infections and sequelae in Östersund [39]. Secondly, outbreaks have been also caused by Cryptosporidium species that were not considered previously to be important human pathogens. Cryptosporidium cuniculus, a parasite of rabbits, caused a waterborne outbreak in Northamptonshire, UK, involving 23 laboratory-confirmed cases and an estimated 422 cases of cryptosporidiosis above baseline [42]. Investigation of this outbreak revealed an unusual distribution of cases in terms of age (lack of cases in children under 5) and sex (girls and women were overrepresented); interestingly, a similar age distribution was found during subsequent investigation of sporadic cases attributed to C. cuniculus [50], suggesting that exposure and risk factors are parasite-specific. Another species, C. meleagridis, was confirmed in one case during an outbreak involving three farm workers at an organic farm in Sweden [38]. This species infects many host classes including birds, mostly farmed poultry, and has a clear zoonotic potential, but it has not been regarded thus far as an important human pathogen in Europe [4]. Thirdly, adverse climatic events, including heavy rainfall and river flooding, are globally on the rise and can cause epidemics of gastroenteritis. For example, an outbreak in August 2013 in the city of Halle, Germany [33], began 6 weeks after the river Saale overflowed the floodplain and parts of the city centre. The outbreak was caused by C. hominis, subtype IbA9G2, suggesting that human waste was the source of water contamination. Analysis of environmental samples showed high levels of oocysts (up to 592 oocysts/100 L), again suggestive of wastewater contamination, but genotyping was unsuccessful. Of the 167 notified cases, most occurred among children, and playing on previously overflowed floodplain was associated with infection. Thus, this event indicated that public health

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No. of laboratory-confirmed cases

No. of estimated cases

Country and location

Year

Month

Setting or vehicle

Species and gp60 subtype

Spain, Granada

2014

September to November

Nursery school

C. hominis IaA11R2 (5) and IbA10G2 (1); three children also had Giardia

7

Unknown

Germany, Halle

2013

August

Playing on floodplain

C. hominis IbA9G2v

167

Unknown

England, Yorkshire

2013

April

Open farm

C. parvum IlaA19G1R1

35

46

Sweden, Uppsala

2013

March

Veterinary students

C. parvum IIaA16G1R1b (four cases) and IIdA24G1 (two cases)

6

13

Finland; various locations Europe

2012

October and November Summer

Frisée salad

C. parvum IIdA17G1

18

>250

Community setting; vehicle not known

C. hominis IbA10G2

>300

2012

England and Scotland

2012

May

Precut mixed salad leaves

C. parvum IIaA15G2R1

1.8- to 4.9-fold increase compared to previous years 74

Sweden

2011

June

Bird contact

C. meleagridis

1

3

Sweden, Skellefteå Sweden, Östersund England, Greater Manchester Norway

2011 2010 2010

May November September

Drinking water Drinking water Swimming pool

C. hominis IbA10G2 C. hominis IbA10G2 C. hominis

Unknown 186 3

~20.000 ~27.000 48

2009 and 2012

March and March

Holiday farm with two outbreaks

C. parvum IlaA19G1R1

11 and 15

55 and 40

England, Northamptonshire

2008

June

Drinking water

C. cuniculus VbA18

23

422

Novel findings or key messages

Reference

Mixed infections may occur indicating multiple possible sources and/or high contamination and/or transmission. Oocyst survival in the environment and adverse climatic events can increase the risk of cryptosporidiosis. Handwashing and provision of information about infection risks are important interventions in these settings. Contact with calves and eating in clinic cars were risk factors, while handwashing was protective. Provision of information about infection risks is important, even for occupationally exposed professional groups. Food trace-back identified possible causative factors for contamination. Lack of routine diagnosis, reporting, species identification and genotyping in some countries made it hard to define the extent of the outbreak. Genotyping was important in case definition; improvements are needed in food trace-back. Outbreak involving a species other than C. hominis or C. parvum; zoonotic source identified. High attack rate and postinfectious sequelae identified. High attack rate and postinfectious sequelae identified. People with cryptosporidiosis continued to swim.

[32]

Varying rates of secondary transmission were reported, from both asymptomatic and symptomatic infections, emphasising the importance of personal hygiene. First outbreak involving a species other than C. parvum or C. hominis. Syndromic surveillance indicated the outbreak was larger than identified from diagnosed cases.

[33] [30] [34]

[35] [9,36] [37]

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TABLE 2. Selected outbreaks of cryptosporidiosis in Europe

[38] [39] [39,40] [41] [11] [42,43]

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authorities should consider the potential health risks of longterm surviving Cryptosporidium oocysts. Finally, foodborne outbreaks of cryptosporidiosis have been recently documented in several EU countries, and implicated foods include fresh produce and unpasteurized or inadequately pasteurized milk [49]. Of particular importance was the outbreak in May 2012 that occurred across England and Scotland [37] (Table 2). To date, this is the largest documented outbreak of cryptosporidiosis attributed to a food vehicle, with >300 individuals involved. Although the precise source was not identified, infection was strongly associated with the consumption of precut mixed salad leaves sold by a single retailer, and typing revealed the outbreak strain to be C. parvum gp60 subtype IIaA15G2R1, a worldwide-distributed subtype highly prevalent in both livestock and humans. Also in 2012 an outbreak occurred in Finland that was nearly as large as that in the United Kingdom, caused by C. parvum IIdA17G1, and was associated with consumption of frisée salad [35]. The traceback investigations revealed the source of the frisée salad as an outdoor grower in the Netherlands, where there had been heavy rainfall during the growing season. In recent years, consumption of raw vegetables has been linked to outbreaks of cryptosporidiosis in Denmark, Sweden and Finland, but this trend is likely the result of good outbreak reporting and indepth case interviews [49]. In the second half of 2012, several countries (including the United Kingdom, Germany, Belgium, Spain, Sweden, Finland and the Netherlands) reported an unusual increase in the number of cases of cryptosporidiosis [9,36]. The annual number of reports to ECDC (excluding nonreporting countries and those where national surveillance systems for cryptosporidiosis reporting do not cover the whole population) was 68% higher than in 2011, with 9591 cases reported, but no common epidemiologic link could be identified [9,36]. Widespread outbreaks raise the need for standardized subtyping schemes for Cryptosporidium. Analysis of outbreaks can provide information for future prevention by identifying the main causes, understanding the risks and ensuring effective interventions are in place. For drinking water, catchment protection measures identified through water safety plans, appropriate treatment (especially the installation of filtration), secondary disinfection such as ultraviolet light, and protection of water in distribution are key interventions [51]. In the United Kingdom, a demonstrable reduction in the number of drinking water outbreaks and cases attributed to mains water has been linked to improvements in water quality through the closure of or installation of filtration at unfiltered supplies [19,52]. Interestingly, one study in Scotland suggested that while oocyst removal from drinking water supplies decreased the risk of waterborne cryptosporidiosis,

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the reduction in circulating antibody response may indicate decreased protective immunity and that the population may therefore be at increased infection risk from exposure to other sources [53]. While outbreaks linked to drinking water have been shown to have declined in the United Kingdom, those linked to recreational waters, especially swimming pools, have not. Similarly, in the United States, Cryptosporidium is the predominant cause of outbreaks associated with recreational waters [54]. The problem of Cryptosporidium resistance to chlorine at normal pool water treatment levels (0.5 to 2.5 mg/L residual free chlorine) means that treatment to deal with contamination relies on good filtration practices, with an option for emergency decontamination by superchlorination (http://pwtag.org/ technicalnotes/superchlorination-of-swimming-pool-water/). Superchlorination, whereby the free chlorine is raised for a sufficient time to provide a 3-log inactivation (20 mg/L for 13 hours for Cryptosporidium), provides no long-term protection, and it must be carried out by correctly trained operators who understand the procedure and that for subsequent dechlorination to normal levels. Adverse effects include the generation of potentially harmful by-products and the corrosive effect on the treatment plant. However, prevention of contamination of pools in the first place is preferable; this requires improved hygiene by pool users, and communication of and adherence to advice to patients not to swim for 2 weeks after symptoms cease. It has been found that in some outbreaks symptomatic people continued to use swimming pools [39]. Communicating risk and appropriate preventative measures has also been found to be important in reducing the risk of infection and outbreaks at petting farms. One outbreak investigation found that lack of verbal advice and noncompliance with handwashing were significantly associated with the risk of acquiring cryptosporidiosis at a petting farm [30]. The need for the provision and reinforcement of similar preventive messages was also identified after an outbreak among veterinary students in Sweden [34]. No major waterborne or foodborne outbreaks of cryptosporidiosis have occurred in some regions of Europe, particularly in Southern Europe, thus implying a different epidemiologic situation. However, this may be related to an absence of notification and a lack of investigation rather than to a truly different epidemiology.

Treatment Treatment options for cryptosporidiosis are still limited. Specific treatment strategies for cryptosporidiosis have been pursued for more than three decades, yet despite the evaluation of

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nearly a thousand chemotherapeutic agents, therapies able to clear the host of Cryptosporidium are lacking [55]. While research on potential drug targets is ongoing and a number of candidates have been identified, also through repurposing of drugs developed for other indications [56], none has advanced to clinical trials. This is of particular concern, considering the benefit that an available treatment will have for risk groups such as young children, the elderly, individuals with compromised Tcell function and patients with primary T-cell deficiencies, such as severe combined immunodeficiency and CD40 ligand deficiency (hyper-IgM syndrome) [57]. The introduction of the highly active antiretroviral therapy (HAART) has greatly reduced the impact of cryptosporidiosis among HIV-infected individuals [58]. This is due to an improvement in CD4 cell count and the restoration of a degree of immunity, but also to a direct effect of protease inhibitors on host cell invasion and parasite development in vitro, an effect enhanced with paromomycin [59]. Unfortunately, HAART is costly and is thus not readily available in poor regions of the world, where HIV/AIDS and opportunistic cryptosporidiosis remains a major health problem [56]. Currently nitazoxanide (NTZ) (Alinia, Romark Laboratories), a thiazolide drug with broad antiparasitic activities, is the only US Food and Drug Administration–approved drug for use against cryptosporidiosis in immunocompetent patients, but it is not licensed in Europe. There is little evidence for efficacy of NTZ in immunocompromised individuals and malnourished children [60]. The lack of efficacious drug treatments indicates that the development of strategies that prevent disease or reduce the severity of infection, including vaccination, remains essential. Progress in this field has been hampered by lack of systems for continuous in vitro culture, cryopreservation of the parasite, adequate animal models and tools for manipulation of the parasite genome. However, a recent study has demonstrated that genetic analysis and manipulation of the parasite is possible, and that stable transgenes are obtained by delivering in vitro transfected sporozoites directly into mouse intestine [61]. The availability of luciferase reporter parasites will permit screening of novel compounds in vitro or in animals, and gene deletion experiments can be used to validate biologic targets. Attenuated parasites may be obtained through genetic modification, potentially leading to the development of an oral vaccine [62]. A better understanding the mechanisms underlying disease and protection, however, remains crucially required to design and produce a vaccine [63]. Furthermore, it needs to be considered that the efficacy of a vaccine may be reduced in very young children, also because of possible interference by maternal antibodies, micronutrient deficiencies and persistent exposure to other pathogens. Likewise, the use of a live,

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attenuated vaccine might be feasible for immunocompetent individuals, but serious complications exist for immunocompromised individuals. The increasing availability of whole genome sequences from different Cryptosporidium species, as well as functional genomics and metabolomics data, will assist in the identification of new drug targets [64].

Conclusions The burden of cryptosporidiosis, including acute infection and long-term sequelae, in Europe is currently unknown. Improved monitoring and surveillance systems are necessary to define the true epidemiologic situation and to ascertain possible differences among countries. Interventions have been identified from epidemiologic and outbreak investigations, and disease reduction can be achieved. Proper risk communication, especially to vulnerable populations, remains important. Treatment options are still limited; no fully effective drug treatment or vaccine is available for Cryptosporidium. The recently demonstrated possibility to knock out genes and introduce markers has opened new ways towards drug discovery and vaccine development. Interest in the development of new therapies for paediatric cryptosporidiosis is growing, as witnessed by the inclusion of Cryptosporidium in the last call for Global Grand Challenges funded by the Bill and Melinda Gates Foundation.

Transparency Declaration All authors report no conflicts of interest relevant to this article.

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