ABSTRACT
Human cryptosporidiosis, a diarrheal disease caused by Cryptosporidium species, has previously been considered rare in Denmark and primarily associated with travel abroad. Cryptosporidium in humans came under national surveillance in 2023. Here, we assess all cases identified in Denmark from 2010 to 2024, presenting the identified species, the trends in time and place, and relate the findings to recent changes in diagnostic methods. After 2021, the number of new cases increased substantially, coinciding with the adoption of gastrointestinal syndromic testing in several local hospitals. During seasonal peaks (August-October), Cryptosporidium was detected in the stool of >2% of patients tested. Infections predominantly occurred in individuals without known comorbidities, and hospitalization rates exceeded 10% in recent years. Co-infections with enteropathogenic bacteria were rare (6%), suggesting that Cryptosporidium alone was the causative agent in the patients. Most cases had no history of travel outside Denmark. Beyond C. parvum (56.9%), and C. hominis (11.3%), species of zoonotic relevance were implicated, e.g., C. mortiferum (2.5%), C. meleagridis (1.7%), C. felis (1.2%) and C. erinacei (0.8%). The transition to high-throughput molecular diagnostic methods and the testing of more patients, including those without recent travel history, has dramatically improved the detection of Cryptosporidium in stool samples in Denmark. Cryptosporidiosis appears to be a common and endemic disease in Denmark. The wide heterogeneity of infecting species suggests a number of transmission routes; these are yet to be uncovered. Cryptosporidiosis should be considered a common gastrointestinal infection by clinicians in Denmark and preventive measures should be prioritized.
KEYWORDS: Surveillance, clinical microbiology, Apicomplexa, diagnosis, outbreak, Scandinavia, cryptosporidiosis, public health microbiology
Introduction
Cryptosporidium is a single-celled intestinal parasite and a significant cause of diarrheal disease worldwide [1,2]. Cryptosporidium infects both humans and a large variety of non-human hosts [3,4]. The infective stage of the parasite, the oocyst, can survive in the environment for months and is highly resistant to disinfectants, including chlorine. The infectious dose is small, and as little as 10 oocysts have been proposed to be sufficient to establish disease in humans [5,6]. In immunocompromised individuals, cryptosporidiosis can be severe and even fatal [7]. In low- and middle-income countries, the parasite is linked to moderate-to-severe diarrhoea in children (particularly toddlers), high mortality rates, and long-term effects, including growth stunting [2,8]. Currently, treatment options for cryptosporidiosis are limited to the broad-spectrum antiparasitic drug nitazoxanide, which is not considered effective in immunocompromised individuals [1,9]. Moreover, no vaccine against Cryptosporidium exists for human use yet [10]. Surveillance, including identification of infection sources and transmission patterns, is therefore key in order to control and prevent cryptosporidiosis in humans.
Historically, Cryptosporidium infections were considered rare in Denmark [11] and primarily linked to foreign travel, with only few minor outbreaks detected among veterinary students handling calves [12], and local foodborne outbreaks [13–15.] Meanwhile, in neighbouring countries, large outbreaks were observed. An example is the 2010 outbreak in Östersund, Sweden, where approximately 27,000 individuals became infected due to contamination of a public water supply [16].
Recent implementation of molecular methods including gastrointestinal syndromic testing assays (multiplex PCR panels enabling simultaneous screening for numerous viruses, bacteria and parasites) with Cryptosporidium in them, and inclusion of cryptosporidiosis in the Danish surveillance system, has led to a change in this perception, identifying Cryptosporidium as an endemic and important enteric pathogen in Denmark, with many sporadic cases [17]. This study describes the incidence of the disease, the species involved, and the role of local implementation of syndromic testing in uncovering the epidemiological landscape of human cryptosporidiosis in Denmark.
Methods
Study setting
Local diagnostics
In Denmark, testing for Cryptosporidium in human clinical faecal samples is performed at the clinical microbiology departments (CMDs) of which there are ten across the country. The methods used for testing vary between CMDs and have also changed over time [11]. Additionally, the criteria for Cryptosporidium testing vary between hospitals. Taken together, this has resulted in regional differences in sample triage procedures and in the ability to identify cases; e.g. while some CMDs use microscopy and only test patients with a history of travel, hospitalization, or symptoms lasting for more than 7 days, others use multiplex PCR panels to test all patients with diarrhoea regardless of medical and exposure history.
National laboratory surveillance
Human cryptosporidiosis was not officially a notifiable disease in Denmark until November 2023. However, national clinical laboratory data are available from the Danish National Microbiology Database (MiBa) [18], which has information on all laboratory reports from Danish CMDs since January 2010. Therefore, data on all laboratory-confirmed cases of cryptosporidiosis from 1 January 2010, to 31 December 2024 were included in this study.
Data from MiBa is standardized using central dynamic mapping of local CMD codes into shared standard coding, which can be used for surveillance purposes and research. This standardized version of MiBa, called EpiMiBa, is a mirrored version of MiBa. Data from EpiMiBa is subsequently used in “Keys to Infectious Disease Surveillance” (KIDS)[19], which is the fully automated disease surveillance datamart of Statens Serum Institut (SSI), and which applies three levels to produce an output for each of the infectious diseases it contains: Level 1) demarcation of all potentially relevant tests, e.g. Cryptosporidium mentioned in test requisitions or results codes; Level 2) interpretation of test results into “positive,” “negative,” or “irrelevant result”; and Level 3) application of an episode definition, where test results are converted to cases and are used to determine reinfections. For Cryptosporidium, KIDS uses a rolling time window of 60 days between two consecutive positive tests to define episodes.
Confirmation and species identification
The CMDs have been submitting samples positive for Cryptosporidium to the national Reference Laboratory for Parasitology at SSI on a voluntary basis since late 2021. At SSI, original faecal material is subject to DNA extraction, where confirmation of Cryptosporidium spp. relies on a positive real-time PCR result [20]. Samples that are real-time PCR-positive are subject to species identification using PCR and Sanger sequencing of the small subunit (SSU) ribosomal RNA (rRNA) and actin genes, using standard procedures previously accounted for [21,22].
Data Sources
Data available from KIDS included sample number, sample date, and the name of the diagnosing CMD (see Supplementary Figure 1 for geographical distribution of CMDs). Over the 15-year study period, variation existed in the way in which CMDs deposited information into MiBa, and some CMDs changed their reporting methods during the study period. As a result, the availability of data – such as name of diagnosing CMD and travel history – was not consistent across all years. Travel information to countries abroad was sometimes included in laboratory test requests from clinicians and, therefore, was also present in MiBa. For samples from 2017 and onwards, this information could be extracted from EpiMiba, whereas information on travel history for samples registered before 2017 was assessed manually. Data on test activity were collected from the four CMDs that implemented syndromic testing (with the QIAstat-Dx® Gastrointestinal Panel [QIAGEN, Hilden, Germany]). Data on age at sampling date and sex were identified using the patients’ unique 10-digit Danish civil registration numbers (CPR numbers). Data from the Danish Civil Registration System (CRS)[23] were used to obtain date on death (data extracted March 2025) for those cases that had deceased. The CRS was also used to obtain information on the home addresses of the cases (at the time of sampling), which was used to map the geographical distribution of cases. Data from the National Patient Register (NPR)[24] were used to assess information on hospitalizations and comorbidities among the cases. The NPR includes data on all hospital contacts taking place, including outpatient visits. Bacterial co-infections were identified using the National Register of Enteric Pathogens, which includes data on all laboratory notifications of Salmonella spp., Campylobacter spp., Yersinia enterocolitica, Shigella spp./Enteroinvasive E. coli (EIEC), Shiga toxin-producing E. coli (STEC), and Clostridioides difficile [25]. Data on population size in Denmark and by municipality per year were obtained from Statistics Denmark [26] and used to calculate incidence rates.
Variables
A new case of cryptosporidiosis was defined as a sample testing positive at least 60 days after the last positive sample (KIDS level 3 episodes definition). In some results, the study period was divided into two periods, “before 2022” and “2022–2024,” because 2022 was the first full year after the local implementation of syndromic testing to identify Cryptosporidium. Age was reported as a continuous variable and stratified into age groups.
For simplicity, the ten Danish CMDs were randomly assigned a label of roman numerals between I and X (Supplementary Figure 1). Samples from CMD I and II before 2018 were registered with the same CMD name in MiBa; therefore, cases from these two CMDs could not be separated in this period and were instead shown together as “CMD I/II” where relevant. The uptake areas of the two CMDs border each other.
A patient was considered hospitalized if admitted to hospital within a 14-day period starting from 7 days before and ending 7 days after the sampling date, regardless of ICD-10 codes. Only hospital stays lasting for longer than 12 hours were considered “hospitalizations.” Comorbidities were identified using ICD-10 codes from the NPR within five years preceding sampling, categorizing cases as immunosuppressed or not (see definition in Supplementary Table 1), and using the Charlson Comorbidity Index (CCI) (described in Supplementary Table 2). Furthermore, using the CCI score, individuals were then grouped into one of three CCI groups: 0 = “none,” 1–2 = “mild,” and ≥3 = “high.” Mortality was assessed as all-cause 30-day mortality. A case was considered co-infected if a laboratory notification of an enteric bacterial pathogen was available with a sampling date within 14 days of a Cryptosporidium-positive sample.
Statistical analyses
All analyses of data and graphical representations were made using R version 4.4.0 [27]. Analyses were primarily descriptive and exploratory. When assessing differences in categorical outcomes between groups, the Fisher’s exact test was used; for continuous outcomes, Welsh’s t-test was used when comparing two groups, and the ANOVA was used when comparing multiple groups or the Wilcoxon and Kruskal Wallis rank-sum tests for non-normal data; results were reported as p-values without adjustment for multiple tests.
Thirty-four cases had replacement CPR-numbers and could not be found in the registers; these were therefore excluded from Table 1, Table 3, and Figure 3.
Table 1.
Characteristics of cases diagnosed with Cryptosporidium infections including demographics and short-term outcomes in the periods of 2010–2021 and 2022–2024.
| 2010–2021 | 2022–2024 | p | |
|---|---|---|---|
| n | 2207 | 2505 | |
| Males, n (%) | 1012 (45.9) | 1135 (45.3) | 0.730 |
| Age, median [IQR] | 31 [20, 46] | 35 [21, 54] | <0.001 |
| Age group, n (%) | <0.001 | ||
| <1 | 7 (0.3) | 8 (0.3) | |
| 1–4 | 205 (9.3) | 191 (7.6) | |
| 5–14 | 191 (8.7) | 209 (8.3) | |
| 15–24 | 358 (16.2) | 372 (14.9) | |
| 25–44 | 843 (38.2) | 805 (32.1) | |
| 45–64 | 483 (21.9) | 631 (25.2) | |
| 65–74 | 89 (4.0) | 182 (7.3) | |
| 75–84 | 24 (1.1) | 96 (3.8) | |
| 84+ | 7 (0.3) | 11 (0.4) | |
| Travel history, n (%) | <0.001 | ||
| Unknown | 1197 (54.2) | 147 (5.9) | |
| Yes | 400 (18.1) | 614 (24.5) | |
| No | 610 (27.6) | 1744 (69.6) | |
| Travel history, n (%) | 400 (39.6) | 614 (26.0) | <0.001 |
| Admission to hospital, n (%) | 175 (7.9) | 272 (10.9) | 0.001 |
| Immunosuppression diagnosis (%) | 130 (5.9) | 131 (5.2) | 0.355 |
| CCI group† (%) | 0.506 | ||
| High | 8 (0.4) | 11 (0.4) | |
| Mild | 141 (6.4) | 141 (5.6) | |
| None | 2058 (93.2) | 2353 (93.9) | |
| Dead within 30 days, n (%) | 2 (0.1) | 6 (0.2) | 0.377 |
| Bacterial co-infection* (%) | 90 (4.1) | 193 (7.7) | <0.001 |
*Bacterial co-infections refer to the detection of Campylobacter coli/jejuni, STEC, Shigella spp./EIEC, Salmonella spp., Clostridioides difficile or Yersinia enterocolitica within 14 days of the detection of Cryptosporidium spp., according to data from the National Register of Enteric Pathogens.
†Charlson Comorbidity Index Group: grouped according to Charlson Comorbidity Index: 0 = None, 1-2 = Mild, 3 and above = High.
Table 3.
Characteristics of cases diagnosed with Cryptosporidium infection in Denmark 2010-2024, stratified by Cryptosporidium species.
| Overall | C. parvum | C. hominis | C. mortiferum | C. meleagridis | Other species | Confirmed* | Not confirmable | |
|---|---|---|---|---|---|---|---|---|
| N | 4712 | 493 | 98 | 22 | 15 | 27 | 109 | 102 |
| Males, n (%) | 2147 (45.6) | 211 (42.8) | 47 (48.0) | 10 (45.5) | 4 (26.7) | 14 (51.9) | 50 (45.9) | 34 (33.3) |
| Age, median [IQR] | 33 [21.00, 50.00] | 37.00 [21.00, 54.00] | 34.50 [16.75, 43.00] | 42.00 [5.25, 58.50] | 26.00 [19.00, 51.50] | 44.00 [24.50, 57.50] | 41.00 [21.00, 57.00] | 44.00 [30.00, 62.75] |
| Age group, n (%) | ||||||||
| < 1 | 15 (0.3) | 2 (0.4) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) |
| 1–4 | 396 (8.4) | 33 (6.7) | 8 (8.2) | 5 (22.7) | 2 (13.3) | 1 (3.7) | 4 (3.7) | 8 (7.8) |
| 5–14 | 400 (8.5) | 46 (9.3) | 14 (14.3) | 4 (18.2) | 0 (0.0) | 3 (11.1) | 15 (13.8) | 1 (1.0) |
| 15–24 | 730 (15.5) | 67 (13.6) | 8 (8.2) | 0 (0.0) | 5 (33.3) | 3 (11.1) | 16 (14.7) | 11 (10.8) |
| 25–44 | 1648 (35.0) | 144 (29.2) | 46 (46.9) | 3 (13.6) | 4 (26.7) | 7 (25.9) | 27 (24.8) | 32 (31.4) |
| 45–64 | 1114 (23.6) | 143 (29.0) | 20 (20.4) | 8 (36.4) | 3 (20.0) | 9 (33.3) | 31 (28.4) | 28 (27.5) |
| 65–74 | 271 (5.8) | 34 (6.9) | 2 (2.0) | 2 (9.1) | 1 (6.7) | 4 (14.8) | 14 (12.8) | 13 (12.7) |
| 75–84 | 120 (2.5) | 22 (4.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.9) | 6 (5.9) |
| v84+ | 18 (0.4) | 2 (0.4) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.9) | 3 (2.9) |
| Hospitalized, n (%) | 447 (9.5) | 71 (14.4) | 5 (5.1) | 1 (4.5) | 1 (6.7) | 2 (7.4) | 9 (8.3) | 14 (13.7) |
| Immunosuppressed, n (%) | 261 (5.5) | 19 (3.9) | 5 (5.1) | 0 (0.0) | 0 (0.0) | 2 (7.4) | 5 (4.6) | 11 (10.8) |
| CCI group, n (%) | ||||||||
| High | 19 (0.4) | 1 (0.2) | 0 (0.0) | 0 (0.0) | 1 (6.7) | 0 (0.0) | 0 (0.0) | 2 (2.0) |
| Mild | 282 (6.0) | 28 (5.7) | 2 (2.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 8 (7.3) | 8 (7.8) |
| None | 4411 (93.6) | 464 (94.1) | 96 (98.0) | 22 (100.0) | 14 (93.3) | 27(100.0) | 101 (92.7) | 92 (90.2) |
| Dead within 30 days, n (%) | 8 (0.2) | 1 (0.2) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.9) | 1 (1.0) |
| Travel history, n (%) | 1014 (30.1) | 69 (14.3) | 41 (43.6) | 3 (13.6) | 8 (61.5) | 7 (30.4) | 34 (31.8) | 22 (22.0) |
| Bacterial co-infection, n (%) | 283 (6.0) | 38 (7.7) | 7 (7.1) | 1 (4.5) | 8 (53.3) | 4 (14.8) | 8 (7.3) | 13 (12.7) |
| Campylobacter coli/jejuni, n (%) | 126 (44.5) | 11 (28.9) | 4 (57.1) | 0 (0.0) | 7 (87.5) | 3 (75.0) | 5 (62.5) | 7 (53.8) |
| Escherichia coli (STEC), n (%) | 62 (21.9) | 14 (36.9) | 0 (0.0) | 1 (100.0) | 0 (0.0) | 1 (25.0) | 0 (0.0) | 2 (15.4) |
| Shigella spp./EIEC, n (%) | 41 (14.5) | 1 (2.6) | 3 (42.9) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (12.5) | 1 (7.7) |
| Salmonella spp., n (%) | 28 (9.9) | 2 (5.3) | 0 (0.0) | 0 (0.0) | 2 (25.0) | 0 (0.0) | 1 (12.5) | 1 (7.7) |
| Yersinia enterocolitica, n (%) | 17 (6.0) | 7 (18.4) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (7.7) |
| Clostridioides difficile, n (%) | 27 (9.5) | 3 (7.9) | 1 (14.3) | 0 (0.0) | 1 (12.5) | 0 (0.0) | 2 (25.0) | 1 (7.7) |
*Confirmed, but species identification not possible.
Figure 3.
Maps of annual incidence of Cryptosporidium infections within each Danish municipality per 100,000 inhabitants, 2010–2024. Municipalities indicated in grey reported no cases.
Results
Incidence
A total of 4,746 cases of cryptosporidiosis were identified during the study period, resulting in a mean incidence rate of 5.43 (95%CI = 2.77–8.10) per 100,000 individuals per year. An increase in incidence rate was observed over time, except during the COVID-19 pandemic years, 2020 and 2021 (Figure 1). Notably, the annual case counts more than doubled in 2022, 2023, and 2024 compared with pre-pandemic levels, peaking at 938 cases (15.8 per 100,000) in 2023.
Figure 1.
The incidence of Cryptosporidium infections per 100,000 inhabitants per year in Denmark from 2010 to 2024.
Demography
We found more women than men with cryptosporidiosis, both before and after 2022 (Table 1). The median age at sample date was lower before 2022 than in 2022–2024, 31 years (IQR = [20, 46]) and 35 years (IQR = [21, 54]) respectively. Most positive cases were observed among the 25–44-year-olds, followed by the 45–64-year-olds. The age groups above 45 were more frequently represented in 2022–2024 compared with previous years. Although children aged 1–4 years accounted for less than 10% of the cases both before and after 2022, they exhibited the highest incidence rate relative to their population size: 7.8 per 100,000 individuals per year in 2010–2021 and 25.4 per 100,000 individuals per year in 2022–2024 (see Supplementary Table 3). For more than half of the cases identified before 2022, no information on travel abroad could be identified. For those with information on travel available, 39.6% and 26.0% of the cases identified before and after 2022, respectively, had travelled outside Denmark (see Supplementary Table 4 for travel destinations related to the different species). Cases requiring admission to hospital increased from 7.9% before 2022 to 10.9% in 2022–2024 (p = 0.001). The percentage of immunosuppressed individuals remained similar in both periods (5.5% overall; p = 0.355). Most cases had none of the comorbidities included in the CCI (93.6% overall), and the distribution within the CCI groups was not significantly different between the two time periods (p = 0.506). All-cause 30-day mortality was low overall (n = 8). In 2022–2024, a larger proportion of cases had a bacterial co-infection (7.7%) than in 2010–2021 (4.1%), p < 0.001.
Trends across CMDs
The trends across the CMDs varied (Figure 2), with four CMDs showing higher case numbers in 2022, 2023, and/or 2024, coinciding with the introduction of syndromic testing assays (indicated by dashed lines in Figure 2). Specifically, these CMDs (I, III, IV, and V) identified most of the cases contributing to the increase after 2021. A seasonality pattern for the entire study period was observed with case numbers peaking in August and remaining higher throughout the latter half of the year than in the beginning of the year, spring, and early summer months (Supplementary Figure 2).
Figure 2.
Number of diagnosed Cryptosporidium infections per month in Denmark, 2010–2024, stratified by clinical microbiology department (CMD). Dashed lines mark the implementation of the QIAstat-Dx Gastrointestinal Panel. Before 2018, samples from CMD I and CMD II were collectively registered under the same CMD ID, thus case numbers from this period are presented as coming from “CMD I/II”
Geographical incidence
During the early years of the study period, cases were identified only in a minority of the 98 Danish municipalities (Figure 3). By 2022, however, the annual incidence rate in certain municipalities in the islands of Zealand and Funen, which constitute the uptake areas of CMD I/II, III and IV (see Supplementary Figure 1 for the geographical distribution of CMDs in Denmark), had risen to 50 cases per 100,000 individuals. In 2023 and 2024, the North Denmark Region (uptake area of CMD V) also began reporting a significant number of cases. In contrast, the Central Denmark Region and Southern Jutland (uptake area of CMD X, VIII and VI) consistently reported few cases, with some municipalities identifying only a handful of cases throughout the entire period from 2010 to 2024.
Test activity and percentage positive
Data on test activity collected from the four CMDs that implemented syndromic testing showed that test activity increased severalfold after the introduction of syndromic testing (Figure 4). Of note, the proportion of tests that were positive remained at a similar level or was slightly higher after the introduction of syndromic testing, peaking during the August – October period at near or above 2%. In the North Denmark Region (uptake area of CMD V, see Supplementary Figure 1), test activity halved around the turn of the year 2023–2024, following a change in sample triage procedure that limited syndromic testing to samples only from hospitalized individuals, children less than seven years, or from those with a recent travel history.
Figure 4.
Number of tests performed to detect Cryptosporidium infections (blue line, left y-axis) and percentage of tests positive for Cryptosporidium (green bars, right y-axis) per month, stratified by clinical microbiology departments (CMDs) in Denmark, 2018–2024. Only data from CMDs that have implemented syndromic testing were included. The dashed lines mark the implementation of syndromic testing assays. The data for CMD V include only numbers for the period of syndromic testing, while the data for the three other CMDs also include numbers from specific PCR-methods used prior to the introduction of syndromic testing.
Species identified
Among the 866 samples sent to SSI for confirmation from 2021 to 2024, 13 species of Cryptosporidium were identified (Table 2). Most of the samples (56.9%) were positive for C. parvum followed by C. hominis (11.3%), C. mortiferum (2.5%) and C. meleagridis (1.7%). A considerable proportion of samples, 12.6%, could be confirmed as Cryptosporidium-positive, but a specific species could not be identified. A further 11.8% of the samples could not be confirmed positive for Cryptosporidium spp.
Table 2.
Samples received for confirmation at the national reference laboratory of parasitology, Denmark, 2021–2024 and Cryptosporidium species identified.
| n | % | |
|---|---|---|
| Samples received for confirmation and species identification | 866 | |
| C. parvum | 493 | 56.9 |
| C. hominis | 98 | 11.3 |
| C. mortiferum | 22 | 2.5 |
| C. meleagridis | 15 | 1.7 |
| C. felis | 10 | 1.2 |
| C. erinacei | 7 | 0.8 |
| C. canis | 4 | 0.5 |
| C. cuniculus | 1 | 0.1 |
| C. ditrichi | 1 | 0.1 |
| C. equi | 1 | 0.1 |
| C. occultus | 1 | 0.1 |
| C. tyzzeri | 1 | 0.1 |
| C. viatorum | 1 | 0.1 |
| Cryptosporidium-specific DNA confirmed but species identification not possible | 109 | 12.6 |
| Cryptosporidium-specific DNA not identified | 102 | 11.8 |
| Not sent for confirmation and typing | 3846 |
Species-specific epidemiology
All species, except for the “Other species,” were most frequent in females, most evidently exemplified by cases with C. meleagridis (73.3%) (Table 3). The median age and interquartile ranges highlighted subtle differences among the species, with the group of cases where Cryptosporidium spp. could not be confirmed and the group with “Other species” skewing older than the rest. C. hominis peaked in middle-aged adults (25–44 years), C. meleagridis peaked in the age group 15–24, C. mortiferum peaked in the 45–64-year-olds but had a notable proportion of children below 15 years of age (>40%), and C. parvum peaked in the groups 25–44 years and 45–64 years. All species showed peaks in August (data not shown). The category of “not sent for confirmation” had a similar distribution per month as the total group of Cryptosporidium-positive samples.
C. parvum was the species with the highest proportion of hospitalized cases (14.4%); the proportion was less than 10% for each of the other species. Regardless of species, most cases had no immunosuppression (94.5%), and comorbidities were rare. Deaths within 30 days were rare overall (n = 8).
Cases of C. parvum and C. mortiferum were predominantly domestically acquired, with only 14.3% and 13.6%, respectively, being associated with a history of travel. In contrast, 61.5% of C. meleagridis cases had a history of travel, while for C. hominis and “Other species” travel history was registered in less than half of the cases; 43.6% and 30.4%, respectively. C. meleagridis cases and cases infected with “Other species” primarily had a history of travel to countries in Asia, while C. parvum, C. hominis, and C. mortiferum primarily had a travel history in European countries (see Supplementary Table 4).
Bacterial co-infections were identified in 6.0% of the cases, with the highest occurrence observed in cases with C. meleagridis infections (53.3%), followed by those that were infected by “Other species” (14.8%). Campylobacter spp. was the most frequent of the co-infections (44.5%), present in all but one C. meleagridis co-infection as well as in over 50% of co-infections involving “Other species” and C. hominis, and 28.9% of those involving C. parvum. STEC was implicated in 21.9% of co-infections, including the sole co-infection with C. mortiferum, and 36.9% of C. parvum co-infections.
Discussion
This study demonstrates a considerable increase in detected cases of cryptosporidiosis in Denmark after 2022. This rise is attributed to advances and changes in diagnostic methods and algorithms. Specifically, it is the introduction of PCR-based syndromic testing assays and the testing of many more patients that has improved the detection and identification of Cryptosporidium. This tendency is similar to what has recently been described in Norway [28,29].
Case characteristics
The demographic data revealed that cases were most commonly observed among the younger and middle-aged adults, while older individuals were notably underrepresented, although increasingly represented after 2022. The highest incidence rates were observed in young children (1–4 years of age). The varying age distribution may reflect factors such as occupational exposure, travel, and differences in immunity, while differential testing activity also plays a role.
Many of the cases identified appeared to be sporadic, and the exact sources of infection remain unclear. Five cases from 2023 were known to be linked to an outbreak of cryptosporidiosis from contaminated kale in Sweden [30], but more cases may well have been part of outbreaks that were not identified.
Geographical and CMD trends
An increase in the incidence rate of Cryptosporidium infection was uncovered across all Danish municipalities within the uptake area of CMDs that routinely employ syndromic testing assays for Cryptosporidium. We found that these CMDs tested many more individuals for etiological agents of diarrhoea (including Cryptosporidium) after syndromic testing was introduced, and that the percentages of positives were as high or higher than those observed when testing methods specifically targeted to identify Cryptosporidium were used. In contrast, CMDs that did not introduce syndromic testing consistently found lower numbers of cases. This suggests that the incidence of cryptosporidiosis was stable during the entire study period, but that a substantial proportion of cases failed to become identified in the early years of the study period. Therefore, the regions of Denmark in which the incidence of Cryptosporidium still appears low may just have a higher burden of unidentified cases. Additionally, our findings indicate that Cryptosporidium is the cause of diarrhoea in at least 2% of cases tested in Denmark during the seasonal peak in August – October, with a similar incidence rate as Salmonella infection [31].
Outcomes and comorbidities
Expanding and increasing testing for Cryptosporidium could have been expected to capture more mild cases of cryptosporidiosis. However, the proportion of hospitalized cases increased from less than 8% before 2022 to over 10% during 2022–2024, now aligning with hospitalization rates reported in other countries [32,33] and comparable to those seen in campylobacteriosis [34]. Although Cryptosporidium infections were associated with hospitalization in approximately 10% of cases, the 30-day all-cause mortality rate remained low across the study period. In Denmark, a high-income country where baseline health generally is good, and the prevalence of population-level health stressors such as malnutrition and inadequately managed underlying conditions are low, Cryptosporidium infections rarely lead to death in the short term.
Most cases had few or none of the comorbidities included in the CCI or in our definition of immunosuppression. The implementation of high-throughput diagnostic techniques therefore highlights that Cryptosporidium is a clinically significant pathogen also among patients without severe comorbidities – prompting them to seek medical care and clinicians to request a screening for intestinal pathogens. Our method for assessing comorbidities holds limitations, as it does not capture comorbidities outside of our predefined criteria; nor does it capture individuals who have not interacted with the secondary or tertiary healthcare system, including those using immunosuppressive drugs without receiving concurrent hospital specialist care. Nevertheless, the method is expected to be able to identify severe comorbidities.
Species identified
C. parvum, the most frequently detected species in Denmark, is considered zoonotic, suggesting that animal contact could play a major role in the transmission of cryptosporidiosis here. Before 2022, only C. parvum, C. hominis and the rare case of C. meleagridis infection were identified [35]. In the more recent years and after the introduction of syndromic testing methods, several additional and also potentially zoonotic species such as C. mortiferum and C. erinacei are being detected at relatively high rates, further supporting the likelihood of animal-to-human transmission of cryptosporidiosis. Specifically, several cases of cryptosporidiosis involving rodent-adapted species have recently been identified in Denmark [21].
The fact that we have identified Cryptosporidium species not previously known to be endemic in Denmark after the introduction of the QIAstat-Dx® Gastrointestinal Panel in Denmark, indicates that this assay can detect a broad range of species. While the rise in cases likely reflects improved detection rather than an actual emergence of the Cryptosporidium, our insight into the causes of human cryptosporidiosis in Denmark is becoming more detailed. Case numbers for some species are still small, but different patterns for different Cryptosporidium species are beginning to appear – indications that they infect distinct demographic groups, exhibit varying patterns of co-infection, and differ in hospitalization rates. These findings underscore the importance of molecular characterization of Cryptosporidium.
A considerable part of samples received at SSI were not confirmable. This could be due to storage conditions, transport time, etc., influencing sample quality and/or differences in sensitivity of the methods used locally and the real-time PCR used at the national reference laboratory at SSI. Additionally, a similar proportion of samples could be confirmed, but a species not identified. The reasons for these discrepancies will be subject to further investigations.
Travel
Around 70% of cases with travel information available had no history of travel, so these individuals had likely been infected in Denmark. A recent study from Norway reported a comparable proportion of domestically acquired cases [29]. Especially infections with C. mortiferum and C. parvum appeared to be autochthonous. The seasonal peaks observed during summer and early autumn could be explained, in part, by increased travel activity in this period, and it also aligns with findings in previous studies from other regions of the world, where cryptosporidiosis rates often rise during warmer months, potentially due to factors such as increased outdoor activities – which might increase exposure to animals – and bathing in contaminated recreational water bodies [36].
Co-infections
Co-infections with enteropathogenic bacteria under surveillance were identified in 6% of the cases. We did not have access to data on other enteropathogenic bacteria, parasites, or viruses, but this would undoubtedly have increased the number of co-infections, particularly virus in young children [17]. Co-infecting bacteria were especially Campylobacter and to a lesser extent STEC. Notably, almost half of C. meleagridis cases also tested positive for Campylobacter within 14 days. C. meleagridis has a wide host range but is known to infect both avian and mammalian species, and was first discovered in turkeys [37]. Campylobacter is the most frequently identified bacterial cause of enterocolitis in Denmark and largely attributable to the handling/consumption of undercooked poultry, thus supporting the hypothesis that C. meleagridis may also be transmitted through food/drink or recreational exposure to water contaminated with faeces from birds.
The identification of two (or more) gut pathogens in the same individual within a short time frame raises important questions about whether Cryptosporidium actually was the cause of disease, whether the other pathogen was the cause, or whether simultaneous presence of both pathogens resulted in more severe symptoms. Because more than 90% of cases did not have one of the enteropathogenic bacteria identified in their stool, Cryptosporidium itself was likely to have caused their symptoms. Further investigation into co-infections with Cryptosporidium could provide critical insights into the exposures and environmental factors driving the emergence of C. meleagridis and other Cryptosporidium species, as well as the clinical implications of infections involving more than one pathogen.
Conclusions
The transition to sensitive molecular diagnostic methods, coupled with increasing testing volumes, has enabled the identification of a large number of cases of cryptosporidiosis in Denmark that would previously have gone undetected. We now recognize Cryptosporidium as a significant endemic pathogen in Denmark that causes disease in immunocompetent individuals, some of whom require hospitalization. Cryptosporidiosis in people with diarrhoea in Denmark may be caused not only by C. parvum and C. hominis, but also by a variety of other species, with possible zoonotic transmission from larger mammals, but also from birds and rodents. With the diagnostic improvements and typing methods in place, we will now be able to look into risk factors for infection, pinpoint transmission paths, investigate acute clinical manifestations linked to distinct species, and optimally, any long-term consequences of cryptosporidiosis.
Supplementary Material
Acknowledgements
The authors thank all the Danish clinical microbiology departments for their dedication to optimizing and ensuring high-quality diagnostic methodologies and contributing data to national surveillance. We also extend gratitude for their efforts in providing samples of Cryptosporidium spp. to the reference laboratory for national surveillance.
Funding Statement
The work was supported by the Independent Research Fund Denmark [grant number 3165-00266B].
Disclosure statement
No potential conflict of interest was reported by the authors.
Ethics
This article was prepared on the basis of national surveillance activities that are part of the advisory tasks of SSI for the Danish Ministry of Health. According to section 222 of the Danish Health Act, SSI's purpose is to monitor and fight the spread of disease, and the national surveillance activities conducted by SSI do not require approval from an ethics committee.
Supplemental Material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2025.2529893
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