In November 2010, 27,000 (45%) inhabitants of  stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized  by a rapid onset and high attack rate, especially among  young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily,  and gluten intolerance were identified as risk factors for  acquiring cryptosporidiosis. Also, chronic intestinal disease  and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype  IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can  have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals  (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is  transmitted mainly by the fecal-oral route, usually through   oocyst-contaminated water or food or by direct contact  with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent,   requiring only a few oocysts to establish infection (1,4). In  healthy persons, gastrointestinal symptoms usually resolve  spontaneously within 12 weeks, although asymptomatic  carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term  effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely  unknown, although the disease was recently identified as  one of the major causes of moderate to severe diarrhea in  children <5 years of age in low-income countries (7). In  Sweden, cryptosporidiosis has been a notifiable disease  since 2004, and 150 cases (1.7/100,000 population/year)  were reported annually until 2009. However, cryptosporidiosis is probably underreported, mainly because sampling  from patients with gastrointestinal symptoms and requests  for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health  implications for drinking water and recreational waters.  The oocysts are excreted in large numbers in feces, can  survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to  treat drinking water (9). The first reported outbreak of waterborne human cryptosporidiosis occurred in the United  States in 1984 (10), and since then, numerous outbreaks  involving up to hundreds of persons have been identified  in several parts of the world (11,12). Large Outbreak of   Cryptosporidium hominis Infection  Transmitted through the   Public Water Supply. Cryptosporidium spp.  are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in  Cryptosporidium infections, particularly by C. hominis  IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving  Cryptosporidium has been recognized (Y. Andersson, pers.  comm.), and a C. parvum outbreak associated with fecal  contamination of a public swimming pool occurred in 2002  and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from  194 patients in Sweden during 20062008 identified 111 C.  parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad,  and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in  raw water from 7 large WTPs in Sweden (not including the  WTP of interest in the present study) identified 23 (11.5%)  of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and  has a population of 60,000. The major WTP in stersund  (WTP-) draws surface water from nearby Lake Storsjn  and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported  here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by  rapid sand filtering and chloramination. WTP- is situated  4 km upstream from the major wastewater treatment plant  (WWTP-) to ensure that the drinking water intake will  not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in  stersund received reports from several employers that  10%-20% of employees had gastroenteritis. The office  advised that patients with acute gastroenteritis be tested  for bacterial, viral, and protozoan pathogens. Among 20  patients from whom samples were obtained, 14 cases of  cryptosporidiosis were detected on November 26. The  local health advice line received numerous calls from  persons with gastroenteritis, most of whom lived within  the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on  November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical  characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who  582  Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn,  showing water currents (arrows)  and locations of wastewater  treatment plant, water treatment  plant,  and  contaminating  stream during Cryptosporidium  infection outbreak, stersund,  Sweden, 20102011.  C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide  information about day of onset, home address, and recent  food intake.  Written Questionnaire Two months after the outbreak began, we conducted  a retrospective cohort study, which included a random  sample of 1,524 persons living in stersund, to assess the  extent of the outbreak, clinical characteristics of infected  persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by  assuming a 50% attack rate and a 70% response rate when  calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water  consumption, underlying diseases, and whether the WTP-  supplied water to the persons workplace. Residential WTP  supply was ascertained through population registers. Parents or guardians were asked to respond for children <15  years of age. A case-patient was defined as a person who  lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset  after November 1, 2010, and before January 31, 2011. The  study was approved by the Research Ethics Committee of  the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011,  fecal samples from inhabitants of stersund who had  acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard  methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water,  raw water, and wastewater were collected to trace the  source and monitor the presence of oocysts. Most water  samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions,  sampling was also conducted at 14 other WTPs and 6  additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may  have contaminated the raw water, and samples from those  streams were analyzed for Cryptosporidium. Also, as part  of a then-ongoing national survey regarding presence  of parasites in wastewater, 7 preoutbreak samples were   collected at WWTP-. The methods used are described  in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species  were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species  were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22).  Oocysts in wastewater and stream water samples were  isolated from the contaminating debris by immunomagnetic  separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that  had been obtained from 1 raw water sample and 1 drinking  water sample by use of Envirochek filters (Pall Life Science,  Ann Arbor, MI, USA) followed by IMS. Microscope slides  containing 113 oocysts from 4 raw water samples and 4  drinking water samples were sent to the Cryptosporidium  Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations  between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by   WTP-. Student t test was used to analyze differences in  attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable  were investigated by logistic regression. For dichotomous  predictors, odds ratios were used to measure associations  between clinical cryptosporidiosis and risk factors. Because  of overdispersion in the data, negative binomial regression  was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses  of water consumed per day were evaluated as continuous  variables. All statistical analyses were performed by using  SPSS software version 19 (SPSS Inc., Chicago, IL, USA).  A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653  persons over a period of 2.5 weeks, confirming the large  outbreak in the city and contamination of the drinking  water (Figure 2). The number of cases of gastrointestinal  illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day  rapidly declined.   Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 20, No. 4, April 2014  583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to  respond. Of the remaining 1,508, a total of 1,044 (69.2%)  responded: 481 men (46.1%) and 563 women (53.9%)  (median age 44 years Diarrhea[range 098 years])(Table 1). The  response rate was highest for women 6069 years of age  (90.0%) and lowest for men 2029 years (43.8%), and  45.2% (95% CI 42.1%48.3%) of all the responders met  the case definition criteria. When the rate of 45.2% was  applied to the total population of stersund (59,500),  results indicated that 27,000 (95% CI 25,04928,738)  inhabitants contracted clinical cryptosporidiosis during  the survey period. The attack rate decreased with age  (p<0.0001; Table 1, Figure 3), was highest (58.0%) for  persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men  and women. The attack rate was 52.2% for respondents  who lived and worked in areas served by the WTP- but  only 12.8% for inhabitants of stersund who neither lived  nor worked in areas served by that plant (p<0.0001; data  not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%),  watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%)  (Table 2). Diarrhea lasted a median of 4 days (range 151  days). Duration of diarrhea decreased significantly with  age (p<0.0001; Table 3, Figure 3), as did the incidence of  584  Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants;  dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak,  stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not  shown). Recurrence of diarrhea after >2 days of normal  stools (defined as a relapse) was reported in 49.1% of the  cases, and >1 relapse occurred significantly more often  among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant  risks for Cryptosporidium infection (Table 3). Chronic  intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and  young age were significantly associated with more days  with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the  national surveillance system: 149 in Jmtland County and  37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence  has been deposited into GenBank under accession no.  KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative  for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking  water and raw water samples collected at the WTP-  on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw  water from Lake Storsjn over the next 2 months (Table  5). The highest number of oocysts in drinking water (1.4  presumptive oocysts/10 L) was detected on December  12, 2010 (online Technical Appendix Figure 1. During  the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in  samples from the distribution network. Densities in raw  water samples were generally higher: 0.23.1 oocysts/  10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on  November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels  from December 31 onward (online Technical Appendix  Figure 2). Oocysts were detected in 4 of 22 raw water samples  from other municipalities near Lake Storsjn but in only  1 drinking water sample from a WTP (online Technical  Appendix Table). All samples of untreated wastewater,  most samples of treated wastewater (11/18), and samples  from recipient water bodies (6/9) contained oocysts. Two  of the 4 investigated streams connected to Lake Storsjn    Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 20, No. 4, April 2014  585 Figure 3. Percentage of ill persons (A) and mean duration of  symptoms fulfilling the case definition (B), stratified by age group  during Cryptosporidium infection outbreak, stersund, Sweden,  20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund,  Sweden, 2010-2011  Symptom  No. positive answers/total no. respondents (%)*  All respondents, N = 972  Case-patients, n = 434  Noncase-patients, n = 538  Diarrhea, >3 stools/d  382/967 (39.5)  382/429 (89.0)  0/538 (0)  Watery diarrhea  343/945 (36.3)  343/407 (84.3)  0/538 (0)  Abdominal cramps  382/952 (40.1)  328/416 (78.8)  54/536 (10.1)  Fatigue  342/921 (37.1)  302/413 (73.1)  40/508 (7.9)  Nausea  301/931 (32.3)  253/396 (63.9)  48/535 (9.0)  Headache  267/920 (29.0)  232/406 (57.1)  35/514 (6.8)  Fever >38.0C  128/909 (14.1)  121/393 (30.8)  7/516 (1.4)  Muscle or joint aches  95/875 (10.9)  80/366 (21.9)  15/509 (2.9)  Vomiting  89/894 (10.0)  76/357 (21.3)  13/537 (2.4)  Eye pain  81/877 (9.2)  67/367 (18.3)  14/510 (2.7)  Bloody diarrhea  16/883 (1.8)  15/345 (4.3)  1/538 (0.2)  *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3  episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010.  Results on the basis of answers from 972 of 1,044 respondents.     RESEARCH contained oocysts (Table 5). The stream closest to WTP-  (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on  November 30 and December 2, respectively; this finding  could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C.  hominis was determined by restriction fragment length  polymorphism and sequence analysis. Subtyping was not  possible because amplification of the gp60 gene failed.  Also, despite repeated attempts, we were unable to amplify  any DNA sequences from oocysts detected in raw water  and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from  the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland  County. No other Cryptosporidium species or subtypes  were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium  infection affecting at least 27,000 inhabitants of stersund,  Sweden, which represents the largest known outbreak in  Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking  water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water,  to our knowledge, this is the first time this pathogen has  been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak.  First, before the outbreak was recognized, alert staff at  the county laboratory suspected oocysts in wet smears of  unstained, concentrated fecal specimens and subsequently  confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had  not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to  be crucial for the decision to issue a boil-water advisory.  Third, the electronic survey was a valuable tool for daily  monitoring of the epidemic curve and evaluating the effect  of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and  website questionnaires in early detection and monitoring of  an outbreak (23,24). The distribution of symptoms among case-patients  with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding  muscle or joint aches, which were reported less frequently  in stersund. Moreover, the median duration of diarrhea,  the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed,  and number of infected family members as risk factors,  which agrees with results from other studies (26,27).  Also, gluten intolerance remained a risk factor after we  controlled for age, sex, and residence in the WTP area,  but this analysis was based on information from only 17  persons and hence should be interpreted with caution. The  mechanism by which gluten intolerance might constitute  a risk factor for cryptosporidiosis is unknown. Duration  of diarrhea was significantly associated with young age  and chronic intestinal disease. Exacerbation of IBD in  cryptosporidiosis patients has been documented (28), and  Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD  (29). C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly  virulent; is excreted in high numbers in feces (1,31,32);  and is the most commonly identified subtype in waterborne  cryptosporidiosis outbreaks, including that in Milwaukee  (3,30,33,34). These characteristics, along with occurrence  of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure,  contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the  high attack rate, even if the low recovery rate is taken into  account. Densities may have been higher at the onset of the  outbreak because of a surge of oocysts in the inlet before  sampling, and secondary household transmission could  have contributed to some of the cases. However, similar  low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required  analysis of at least 100 L of water to identify the low level  of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute  phase of the stersund outbreak, which underscores the  uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of  oocysts (38). Moreover, no reliable assays to test viability  and infectivity of oocysts are available (1). Because only C. hominis IbA10G2 was identified in environmental samples, we  suggest that the outbreak was caused by a single common  source of contamination, although this hypothesis could  not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has several plausible explations. To our knowledge, no posttreatment  contamination or extensive failures in the treatment processes  occurred, and routine tests of the drinking water showed no  increased levels of fecal indicator bacteria. The WTP- had  2 microbiological barriers (ozonation and chloramination) as  recommended by the drinking water regulations in Sweden  for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts  in the raw water. The long-term solution to reduce infective  parasites in stersund was to install a UV water disinfection  system, which was done after the outbreak in December 2010.  In addition, pipes were repeatedly flushed, and and further  sampling was conducted to verify that no potentially viable  oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an  outbreak (40). 
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