**4. Discussion**

In the present study, performed between January 2013 and December 2014 and including samples from 398 patients with cryptosporidiosis, a high diversity of *Cryptosporidium* species and subtypes was identified. *Cryptosporidium parvum* was still the dominant species, and even fewer *C. hominis* cases were identified compared with a previous study performed in the Stockholm metropolitan area between April 2006 and November 2008. Meanwhile, the total number of patients infected with non-*hominis* and non-*parvum* species was quite high (8%), corroborating earlier observations [9].

Species determination was successful for 95% of the samples. *Cryptosporidium parvum* (79%) dominated both in patients infected in Sweden (84%) and abroad (57%), while *C. hominis* was much less common (13%) and identified in only 3% of the domestic cases and in 30% of the cases infected abroad. Mixed *C. hominis* and *C. parvum* infection was observed only in 1 patient, and for 10 of the patients infected with *C. parvum*, the origin of infection was unknown or uncertain. The high occurrence of *C. parvum* compared with *C. hominis* observed in Sweden is similar to the situation in other industrial countries, such as France, Ireland, and Canada [34–36], although a higher percentage of *C. hominis* has been observed in Spain and Australia [37,38]. Shifting trends have been seen over time in the Netherlands and New Zealand, showing the importance of longitudinal studies [39,40].

In terms of the analysis of cases according to age, the observed bimodal distribution (Figure 1) supports previous observations [9], with a relatively high number of cases observed among infants and toddlers and with a second peak—and the largest one—in the 30–44-year-olds. Quite similar age distributions have been observed in studies from Denmark, France, and Canada, but with the second peak in the 20–35-year-olds [34,36,41]. The bimodal age distribution may reflect transmission between parents and their children; however, no such family clusters were detected during the present study. There was a slight difference in the proportions of female and male cases: 55% and 45%, respectively. Interestingly, this difference in gender distribution has been seen in cryptosporidiosis cases in Sweden every year since 2005 (57% and 43% on average for women and men, respectively) [5]. A similar gender distribution was observed in neighboring Denmark in 2010–2014 [41]. Whether this difference in gender distribution reflects a higher awareness among females of the need to seek medical care, or something else, remains unknown.

A few *C. parvum* outbreaks were included in the present study (Table 5), but even if those patients (*n* = 22) are excluded from the dataset, *C. parvum* remains the dominant species in patients infected in Sweden, at least in the four areas that contributed most of the samples (Table 1). Differences in the geographical distribution of *C. parvum* and *C. hominis*, with more *C. parvum* in rural regions and more *C. hominis* in urban settings, have been described [42,43], and a similar tendency was seen in our study, where the rural region of Halland had relatively more cases of *C. parvum* and fewer cases of *C. hominis* compared with the metropolitan region of Stockholm (Table 1). These differences are often attributed to the closer contact with farm animals in rural areas and more traveling activity for people living in urban areas [44].

The subtyping of *C. parvum* showed a great variability; nine subtype families and 42 subtypes were observed. Two *C. parvum* subtype families, IIa (*n* = 164) and IId (*n* = 118), dominated both in patients infected in Sweden and abroad, while the other subtype families (IIc, IIe, IIl, IIn, IIr, IIs, IIt) were found only in one or two isolates each. The most common *C. parvum* subtype in the present study was IIaA16G1R1b (EU647727), observed in 39 patients. Detected in 24 sporadic cases and 4 cases related to an outbreak among veterinary students, it was also previously observed as the most common subtype amongst people infected in Sweden [9,27] (Table 5). This subtype is common in Swedish cattle and has been involved in earlier outbreaks and family clusters in Sweden [9,45], but was not reported amongst 48 subtyped *C. parvum* outbreaks between 2009 and 2017 in the UK [46].

Subtype IIaA15G2R1 is the dominating *C. parvum* subtype in many countries, both in cattle and in humans, and is responsible for numerous outbreaks of human cryptosporidiosis, probably due to its biological fitness and high virulence [41,46–49]. This subtype was found in 33 patients, of whom 16 were infected in Sweden; 13 were sporadic cases, and 3 cases were part of a family cluster (Table 5). This subtype has been detected in a few Swedish calves [50], but has not been associated with any known larger *Cryptosporidium* outbreak in Sweden.

Many of the IIa and IId subtype sequences in the present study exhibited examples of polymorphism in the non-repetitive part, which is sometimes indicated by a lower-case letter when sequences are reported to GenBank [33], but this is not always the case. To overcome this dilemma, all subtypes and subtype variants in Tables 3 and 4 are referred to via a specific GenBank acc. no. As an example, the most common IId subtype, IIdA22G1 (*n* = 37), occurred in three variants in this study, and one of them (AY166806) (*n* = 15) was only detected in domestic cases, most of them in connection to a foodborne outbreak, in which parsley was the suspected vehicle (Table 5). Another variant of this subtype, IIdA22G1c (FJ917374), was identified in 19 cases, of which 15 represented sporadic cases infected in Sweden. Both these variants of IIdA22G1 have been seen in earlier Swedish studies of cattle and humans [9,50]. The third variant of this subtype, KR349104, was seen in only two cases; one infected in France and one in Sweden.

Subtype IIdA24G1 gained attention in 2011 in connection to a foodborne outbreak linking two Swedish cities, and it was also involved in an outbreak in 2013 among veterinary students occurring during the present study [12,27]. The same subtype, which is considered to be quite rare, was linked to 1 out of 48 *C. parvum* outbreaks in the UK where *gp60* subtyping was performed [46].

The subtypes mentioned above, IIdA22G1c and IIdA24G1, were involved in two foodborne outbreaks, which occurred simultaneously in late 2019 in 10 and 12 Swedish counties, respectively. Subtype IIdA22G1c was identified in 122 cases and IIdA24G1 in 86 cases. Spinach juice was the suspected vehicle for subtype IIdA22G1c, while no specific food item could be identified as a source of infection for IIdA24G1 [51].

Two new *C. parvum* subtypes were identified within the subtype families IIe and IIn. Subtype IIeA13G1 (KU852716) was found in a patient infected in Sweden, and subtype IInA10 (KU852717) in two patients; one infected in Tanzania, and one with unclear origin of infection (either Tanzania or Sweden (secondary infection)). *Cryptosporidium parvum* IIe is a well-known anthroponotic subtype family, but since only two cases (both humans from India) have been reported from subtype family IIn [52], it is impossible to say whether this one might be an anthroponotic or zoonotic subtype family. The same is true for the 3 new *C. parvum* subtype families, IIr, IIs and IIt, that were added to the 16 families already described [48]. Subtype IIrA5G1 and IIsA14G1 were found in patients infected in Sweden. Interestingly, a patient with subtype IIsA10G1 was recently described in a publication from Zambia [53]. The patient with subtype IItA13R1 was infected in Tanzania.

One of the objectives of the present study was to investigate whether *C. hominis* subtype IbA10G2 had proliferated in Sweden after the large waterborne *C. hominis* outbreaks in Östersund and Skellefteå in 2010–2011, wherein an estimated 45,500 persons developed cryptosporidiosis. In the earlier study by Insulander et al. [9], performed from 2006 to 2008, 12 of the 17 *C. hominis* samples from patients infected in the Stockholm area carried subtype IbA10G2, while in the present study only two of the eight *C. hominis* patients infected in different parts of Sweden carried this subtype. The higher frequency of this subtype in the earlier study could be explained by the fact that several outbreaks at day care centers, with an index person infected abroad, appeared during that study period, while no such outbreaks were recorded in the current study. Few samples were submitted from the former outbreak areas; four from Jämtland County (Östersund) and four from Västerbotten County (Skellefteå), with all cases representing *C. parvum* (Table 1). The number of reported cases from these two counties during 2013 and 2014 was also low, at eight and five, respectively, and remained quite low during the following years up till now [5]. Therefore, even with these limitations in mind, we speculate that subtype IbA10G2 has failed to establish itself in Sweden following the large outbreaks.

In the US, where IbA10G2 used to be the most common outbreak-related subtype, a new *C. hominis* subtype, IaA28R4, emerged in 2007 as a major subtype in sporadic cases and waterborne outbreaks [54]. Since 2013, subtype IfA12G1R5 has emerged as the dominant *C. hominis* subtype in the US, as well as in Western Australia [37,55]. In the present study, IaA28R4 was observed in two patients, both of whom had recently visited the US, and subtype IfA12G1R5 in two patients, one of whom was infected in Sweden and one in Germany.

*Cryptosporidium hominis* is generally regarded as an anthroponotic species only occasionally infecting other animals. However, recent studies have shown that for a certain subtype family, Ik, equines are natural hosts [56]. During the present study, the first human cases infected with this subtype family, Ik, were detected [29] (Table 4). Two unrelated patients, both infected in Sweden, carried the same *gp60* subtype, IkA18G1. This subtype was recently detected in a horse from China (MK770627)—information reinforcing the suspicion that zoonotic transmission might have occurred. Another uncommon *C. hominis* subtype family, Ii, was found in a father and son after returning from Thailand, where they visited a monkey farm. This *C. hominis* variant was previously described as *C. hominis* monkey genotype, and has rarely been detected in humans [29].

In the present study, 10 *Cryptosporidium* species/genotypes were found in addition to *C. parvum* and *C. hominis*, corresponding to 8% (30/379) of the total number of genotyped samples. This agrees with an earlier Swedish investigation performed on *Cryptosporidium* patients from the Stockholm metropolitan area, where 9% (17/194) of the genotyped samples represented species other than *C. hominis* and *C. parvum*. However, in the Stockholm study, the majority of these infections were acquired abroad; only three patients, two with *Cryptosporidium* chipmunk genotype I and one with *C. felis*, were infected in Sweden [9]. In the present study, an equal number of patients with species other than *C. hominis* or *C. parvum* were infected in Sweden (*n* = 15) and abroad (*n* = 15) (Table 1). This difference might reflect differences in study populations; in the first study, most patients originated from an urban area, while in the present study, many of the patients were from rural areas and probably more exposed to zoonotic *Cryptosporidium* species endemic to Sweden.

The most common cause of non-*hominis* and non-*parvum* infections acquired in Sweden was *Cryptosporidium* chipmunk genotype I, which was diagnosed in five adults; four women and one man. Cryptosporidiosis caused by the chipmunk genotype is considered an emerging infection in the US [19,43]. Meanwhile, in Europe, only one human case (from France) has been reported outside Sweden [19]. The first Swedish cases were diagnosed in September 2007 and August 2008, at a time when no *gp60* subtype method was available for the chipmunk genotype I [57]. However, later analyses have shown that they carried the same subtype as the patients from the present study, XIVaA20G2T1, a subtype that recently was detected in red squirrels in Sweden (unpublished information). Red squirrels are consequently the most possible source of infection for our patients; the other described host animals—chipmunks, grey squirrels, and deer mice— are not native to the country [19,58]. Recent observations have shown a rising number of chipmunk genotype I infections acquired in Sweden, reflecting an emerging infection in this country (manuscript submitted).

*Cryptosporidium erinacei* was first described in European hedgehogs, but has also been identified in other animals, such as horses and rats [59–61]. Most human cases have been reported from New Zealand (*n* = 13), while reports from Europe are scarce, including one case from the Czech Republic and one from France [40,62,63]. The European hedgehog is considered an endangered species in Sweden and other parts of Europe, which might explain the low number of reports from Europe compared with New Zealand, where it has become an invasive species since its introduction about 150 years ago. The two cases found during the present study are the first human *C. erinacei* cases reported from Sweden. The patient infected in Sweden carried a unique *gp60* subtype, XIIIaA23R12, while the patient infected in Greece carried subtype XIIIaA24R9, which has recently been reported in New Zealand [40].

*Cryptosporidium cuniculus* is a common species among rabbits worldwide, while human infection with this species has gained special attention in the UK, where, in addition to a documented outbreak, sporadic cases are quite common [64]. Reports of human infections from other parts of the world are increasing, and sporadic cases have been described in Nigeria, France, Spain, Australia, and New Zealand [40,48]. Two *C. cuniculus* subtype families have been described, Va and Vb. Four of the study patients were infected with Vb, which is the most commonly reported subtype family both in rabbits and humans, as

well as in a few other animals, such as kangaroo and alpaca [65,66]. Subtype family Va, which was observed in one of the patients infected in Sweden, appears to be less frequent globally, and previous human infections were all reported in the UK [64]. These are the first documented human cases of *C. cuniculus* infection in Sweden.

One patient was infected with *C. viatorum* while visiting Kenya [22]. This species was initially thought to be anthroponotic because only human cases had been detected until 2018, when the first non-human host, an Australian swamp rat, was identified [67,68]. This swamp rat isolate was genetically quite different from all known human isolates, but recent studies from China focusing on different rat species have found *C. viatorum* isolates genetically similar to human isolates, and subtyping with *gp60* has demonstrated the same subtype, XVaA3g, in wild rats from China and a human from Australia [37,69].

Another visitor to Kenya was diagnosed with *Cryptosporidium* horse genotype. All horse and donkey samples positive for this species when sequencing of the *gp60* locus was performed carried subtype family VIa [70,71], which has also been found in one human case from Poland (MK784560) (unpublished). Only four cases with subtype family VIb have been described: two human cases, one in the UK and one in the US [54,72], and two cases in four-toed hedgehogs from Japan [73,74]. No reports of the VIb subtype family in equine hosts are available. The patient from our study carried a new *gp60* subtype family (VIc) of *Cryptosporidium* horse genotype. Since no other reports of this subtype family have been published to date, we cannot speculate on how this patient contracted the infection.

Two children of 3 and 5 years of age, respectively, were infected in Sweden with *C. ubiquitum*, *gp60* subtype XIIa-1. These cases were unrelated, and no probable source of infection was identified. *Cryptosporidium ubiquitum* has a wide host range and has been found in wild and domestic ruminants, rodents, and primates, including humans. In the UK, it is believed that most human infections with *C. ubiquitum* are related to exposure to sheep, which are common carriers of subtype XIIa, while infected humans in the US mainly carry the rodent-associated subtypes XIIb and XIIc [20]. The only published *C. ubiquitum* case amongst Swedish animals is from a calf [45], and the occurrence in other animals in Sweden, including sheep, remains unknown and should be investigated.

The natural hosts of *C. suis* are domesticated pigs and wild boars. Human infections are not frequently diagnosed; only around 12 cases were reported prior to our case, which was a two 2-year-old adopted child diagnosed with cryptosporidiosis upon arrival in Sweden from Lithuania [48,75].

Human infection with the feline parasite *C. felis* is not uncommon; indeed, it is regarded as one of the five most common *Cryptosporidium* species infecting humans worldwide [48]. During the study period, four *C. felis* cases were detected, with three patients being infected in Sweden and one in Indonesia. Two of the Swedish patients had known contact with cats, and analyses of fecal samples from cats and their owners using a newly described *gp60* method showed that they shared *gp60* subtypes; thus, zoonotic infection was confirmed [21,30].

One study patient was infected with *C. ditrichi*, a recently described *Cryptosporidium* species in *Apodemus* spp. in Europe [76]. This was the first time *C. ditrichi* was diagnosed in a human, and later on, two more Swedish cases were identified [31].

Eight patients were infected with *C. meleagridis*, the third most common *Cryptosporidium* species infecting humans [77]. *Cryptosporidium meleagridis* has a wide host range, ranging from various birds to humans, and in some countries (e.g., Thailand and Peru), this species is reportedly more common in humans than *C. parvum* [77]. All patients infected with *C. meleagridis* in the present study had acquired infection while traveling in different Asian countries. The same *gp60* subtype was identified in two patients who had made the same journey to China, while the remaining six patients carried different subtypes (Table 6). Subtypes IIIeA17G2R1, IIIeA19G2R1 and IIIeA21G2R1 have been observed in chicken in China, and subtype IIIbA24G1R1 in poultry from Brazil, indicating zoonotic transmission as a possible route of infection [78,79]. Zoonotic transmission has been documented at a

Swedish organic farm, where the infected person and the chickens carried the same *hsp70* and *gp60* subtypes [23,80].

When this study was initiated, the intention was to cover the entire country of Sweden, and all 21 clinical laboratories carrying out parasitology diagnostic tests in Sweden on a routine basis at the time of the study were invited to participate. One limitation is that by the end of the study period, only 12 of the regional laboratories (representing 11 different counties) had provided samples for the study. Our results, however, could very well be considered to reflect the reality, as nine counties only reported zero to one patient each with cryptosporidiosis during the study period (2013 and 2014) [5]. However, the number of referred samples varied considerably between the laboratories, with only four of them accounting for 95% of the submitted samples (Table 1). With this skewed distribution, we cannot conclude that the results of this study might be generalizable to the whole country. Another reason for this biased referral of samples could be the limited access to unfixed stool samples at some laboratories recommending that stool samples for parasitology be fixed in SAF. The intention at the beginning of the study was to avoid receiving samples fixed in SAF or formalin, which has a known negative effect on the PCR success rate [81], but in the second year, one laboratory with a high detection rate of *Cryptosporidium* and limited access to native (i.e., non-preserved) samples was invited to provide fixed specimens. In total, we received 70 samples fixed in SAF, 63 from this laboratory and 7 from other participating laboratories. The success rate of PCR for SAFfixed samples was 80% (56/70), compared with 99% (323/328) for native stool samples. One recent study pointed out the limited availability of native stool material for the molecular analyses of stool parasites [34], but with the increased use of DNA-based diagnostics, the availability of unfixed material has increased, and the problem with fixed samples is in decline.

Another potential bias is that the clinical laboratories performing the primary diagnostics used different testing strategies and detection methods. Some diagnostic real-time PCR methods are designed to primarily detect *C. parvum* and *C. hominis,* and do not target some of the less common and genetically distinct species, which might have led to an underrepresentation of these in some of the counties [82].

#### **5. Conclusions**

*Cryptosporidium parvum* was the dominant species both in cases infected abroad and in domestic cases. The observed occurrence of *C. hominis* was generally low, and no indication of an expansion of the subtype IbA10G2 previously causing the vast *Cryptosporidium* outbreaks in Sweden was found. There was a high diversity of species and subtypes, with 8% of the cases reflecting species other than *C. parvum* and *C. hominis*, some of which were found for the first time in humans in Sweden (e.g., *C. cuniculus, C. erinacei*, and *C. ubiquitum*). Our study also identified humans as a new host of *C. ditrichi*. Overall, zoonotic species and subtypes plays a major role in human cryptosporidiosis in Sweden.

**Author Contributions:** Conceptualization, J.B. and J.W.-K.; methodology, J.B. and M.L.; software J.B. and M.L.; validation, J.B. and M.L.; formal analysis, J.B., J.W.-K. and M.L.; investigation, J.B., J.W.-K. and M.L.; resources, J.B.; data curation, J.B., J.W.-K. and M.L.; writing—original draft preparation, J.B., M.L. and C.R.S.; writing—review and editing, J.B., M.L. and C.R.S.; visualization, J.B., M.L. and C.R.S.; supervision, J.B.; project administration, J.B.; funding acquisition, J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was funded by a grant from the Swedish Civil Contingencies Agency (Grant number: 2012-172).

**Institutional Review Board Statement:** Approval (registration number 2013/201-31/4) was obtained from the Ethical Review Board at Karolinska Institutet, Stockholm, Sweden, including approval to publish the data (date of approval: 27 February 2013).

**Informed Consent Statement:** Patient consent was waived since all samples were from patients with cryptosporidiosis, which is a notifiable disease in Sweden according to the Swedish Disease Act (2004:168) and included in the national surveillance of microbial pathogens. Species identification and genotyping of *Cryptosporidium* and communication of these data form part of the surveillance and do not require consent from the patient (https://www.folkhalsomyndigheten.se/the-public-health-agen cy-of-sweden/communicable-disease-control/surveillance-of-communicable-diseases/notifiable-di seases/ accessed on 24 April 2021).

**Data Availability Statement:** Data presented in this study are available on request from the corresponding author, Jessica Beser. Due to existing general data protection rules, the data are not publicly available.

**Acknowledgments:** The authors are grateful to all the clinical microbiological laboratories that provided samples to the study and the laboratory personnel at PHAS that performed analyses. The authors would also like to thank Margareta Löfdahl for providing epidemiological data and Erik Alm for the development of the software CryptoTyper.

**Conflicts of Interest:** The authors declare no conflict of interest.
