*4.3. Specimen and Data Collection*

Single stool samples (>3 g or >3 mL) were obtained from each participating subject using a sterile sampling cup during the study period at the two hospital settings and stored at 4 ◦C. Collected samples were shipped to the laboratory of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Shanghai, China) without interrupting the cold chain and immediately stored at −70 ◦C until further processing.

A structured questionnaire was used to gather sociodemographic (including age, gender, educational level, occupation, nationality, and place of residence) and clinical (clinical manifestations associated with enteric protist infections, including abdominal distension, diarrhea, lack of appetite, itchy skin, perianal pruritus, constipation, nausea, abdominal pain, number of stools per day and type of stools) data from each recruited patient.

#### *4.4. DNA Extraction and Purification*

Genomic DNA was extracted from each stool sample (0.2 g or 0.2 mL) using a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Purified genomic DNA was stored at −70 ◦C until downstream analysis by polymerase chain reaction (PCR) amplification.

### *4.5. Molecular Detection of Intestinal Protozoan Species*

A direct PCR protocol was used to detect *Blastocystis* sp. [51]. In contrast, nested PCR protocols were used for identifying *Cryptosporidium* spp., *G. duodenalis*, and *E. histolytica* [52–54]. The description of the primer pairs used, the expected size of the obtained amplicons and the cycling conditions of these protocols are provided in Table S1.

## *4.6. Sanger Sequencing Analysis*

Positive-PCR products of the expected sizes were directly sequenced in both directions using appropriate internal primer sets (Table S1). DNA sequencing was conducted at

Sangon Biotech Company (Shanghai, China). Raw sequencing data were viewed using the Chromas Lite version 2.1 sequence analysis program (https://technelysium.com.au/wp/ chromas/. Accessed on 19 May 2021). The BLAST tool (http://blast.ncbi.nlm.nih.gov/ Blast.cgi. Accessed on 19 May 2021) was used to compare nucleotide sequences with sequences retrieved from the NCBI GenBank database. Generated DNA consensus sequences were aligned to appropriate reference sequences using the MEGA 6 software to identify *G. duodenalis* species and assemblages/sub-assemblages. *Blastocystis* sequences were submitted to the *Blastocystis* 18S database (http://pubmlst.org/blastocystis/. Accessed on 19 May 2021) for sub-type confirmation and allele identification. The sequences obtained in this study were deposited in GenBank under accession numbers MW810321-MW810323 (*G. duodenalis*) and MW798733-MW798742 (*Blastocystis* sp.).
