*2.2. Collection of Patient Data*

Each submitted *Cryptosporidium*-positive sample was accompanied by information on the age, sex, and geographical location of the patient. Information concerning travel abroad within two weeks prior to onset of disease and, in some instances, assumed routes of transmission was retrieved from the referral and/or SmiNet and/or the local department of communicable disease control.

#### *2.3. Laboratory Investigations*

The original *Cryptosporidium* diagnoses were made at local clinical laboratories using modified Ziehl–Neelsen staining or real-time PCR. In total, samples from 398 patients from 12 different microbiological laboratories were forwarded to PHAS for molecular analysis (Table 1). In total, 70 of the stool samples had been fixed in sodium acetate–acetic acid–formalin (SAF), while 328 samples consisted of stool without preservative and/or

DNA extracted from unpreserved stool. Most of the SAF-fixed samples had been washed with phosphate buffered saline at the local laboratory before shipment.

**Table 1.** *Cryptosporidium* species in samples provided by 12 participating laboratories across 11 Swedish counties and locally identified as *Cryptosporidium*-positive.


DNA was extracted directly from the stool specimens using a QIAamp DNA mini kit (Qiagen, Germany) according to the manufacturer's recommendations. Prior to extraction, oocysts were disrupted using a bead-beater (Techtum, Sweden). DNA from external laboratories was extracted with local methods prior to submission to PHAS. To characterize *Cryptosporidium* species and subtypes, all samples were initially subjected to nested PCR of the small subunit rRNA (SSU rRNA) gene with subsequent restriction fragment length polymorphism (RFLP) and nested PCR of the 60 kDa glycoprotein (*gp60*) gene followed by sequencing [15–17]. In addition to RFLP analysis, bi-directional Sanger sequencing of the SSU rDNA amplicons was performed on (i) non-*hominis* and non-*parvum* isolates originally identified by RFLP, (ii) new or uncommon *gp60* subtype families, (iii) isolates where no amplification product was obtained at the *gp60* locus, and (iv) isolates where mixed species were suspected by RFLP. Species determination using a 70-kilo Dalton heat shock (*hsp70*) gene segment was performed on a limited number of samples where SSU rRNA PCR failed [18]. For the subtype determination of species not amplified with the Alves *gp60* primers [17], partial sequences of the *gp60* gene were amplified using different PCR protocols, depending on the investigated species, and products obtained by nested PCR were sequenced [19–23].

*Cryptosporidium*-specific actin and heat shock protein (*hsp70*) genes were amplified and sequenced in order to further characterize some uncommon species/genotypes [24,25].

Sequences were edited and analyzed using the BioEdit Sequence Alignment (version 7.0.9.0.) and compared with isolates in the GenBank database using the basic local alignment search tool (BLAST). All chromatograms were manually inspected for the presence of double peaks indicating mixed species/subtypes. In addition, *gp60* chromatograms and fasta files were examined using our in-house *gp60* sequence analyzer software CryptoTyper (unpublished).

Phylogenetic analysis was performed on SSU rDNA and *gp60* DNA sequences generated in the present study as well as sequences from known *Cryptosporidium* species and subtypes. Phylogenetic trees were generated using the neighbor-joining method based on Kimura's 2-parameter model [26]. To estimate robustness, bootstrap proportions were computed after 1000 replications. Evolutionary analyses were conducted in MEGA X (https://www.megasoftware.net/ accessed on 24 April 2021).

Unique and uncommon nucleotide sequences were deposited in GenBank under the following accession numbers: *gp60*—KU727289, KU852701-KU852740; SSU rRNA— KU892559-KU892561, KU892564-KU892566; actin—KU892568, KU892571 and KU892572; *hsp70*—KU892574 and KU892577.

#### **3. Results**

#### *3.1. Participating Laboratories and Patient Demographics*

Samples were provided from 12 of the 21 clinical laboratories performing *Cryptosporidium* diagnostics in Sweden at the time of the study (representing 11 of the 21 counties) (Table 1). Altogether, samples from 398 patients (124 collected in 2013 and 274 in 2014) were received, representing 63% (398/628) of all cases reported in Sweden during these two years [5] (Figure 1). Among the participants, 217 (55%) were women and 181 (45%) were men. The age range was 1 to 88 years (mean (standard deviation), 34.7 (18.09) years); see Figure 1. The majority of the 398 isolates came from sporadic cases (*n* = 369), while 22 were related to outbreaks and seven to family clusters.

**Figure 1.** Number of reported cases of cryptosporidiosis detected in Sweden in 2013 and 2014 according to age group. One case with mixed *C. hominis* and *C. parvum* infection is not included in the figure.
