In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article  The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health  (2017) 17:328  DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health  (2017) 17:328  Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health  (2017) 17:328  Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health  (2017) 17:328  Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health  (2017) 17:328  Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially  from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were investigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Cryptosporidium oocysts but none were detected - apart from the findings in wastewater samples.
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