*2.3. Genetic Characterization of Isolates*

A total of 11 *G. duodenalis* isolates were successfully characterized at the triosephosphate isomerase (*tpi*) locus (Table 1). Sequence analysis allowed identifying assemblages A (90.9%, 10/11) and B (9.1%, 1/11). Further, all 10 assemblage A isolates were assigned to the sub-assemblage AI of the parasite, and the only one assemblage B isolate was identified as sub-assemblage BIV. *Giardia duodenalis* assemblage A was significantly more prevalent in individuals presenting with diarrhea (4/59) than in individuals without clinical manifestations (6/448, chi-squared = 7.37, *p*-value = 0.006; OR = 5.4, 95% CI: 1.5–19.6).

**Table 1.** Diversity, frequency, and molecular features of *Giardia duodenalis* sequences at the *tpi* locus generated in the present study. GenBank accession numbers are provided.


<sup>1</sup> isolates with associated sequences of insufficient quality to clearly determine the presence of single nucleotide polymorphisms.

*Blastocystis* sp. was detected by PCR amplification of the small subunit ribosomal RNA (*ssu* rRNA) gene in 48 samples (Table 2). Sequence analyses revealed the presence of five subtypes (ST) of this protist, with ST3 being the most prevalently found (43.7%, 21/48), followed by ST1 (27.1%, 13/48), ST7 (18.8%, 9/48), ST4 (8.3%, 4/48), and ST2 (2.1%, 1/48). Concerning intra-subtype genetic diversity, alleles 2, 4, and 88 were identified within ST1, allele 9 within ST2, allele 34 within ST3, alleles 42, 92, and 94 within ST4, and alleles 100 and 101 within ST7. No statistical differences in the distribution frequencies of *Blastocystis* STs were observed between diarrheal and non-diarrheal individuals (chi-squared = 3.73, *p*-value = 0.2913).


**Table 2.** Diversity, frequency, and molecular features of *Blastocystis* sp. sequences at the *ssu* rRNA locus generated in the present study. GenBank accession numbers are provided.

#### *2.4. Impact of Coinfection on Diarrheal Symptomatology*

According to our partial least square (PLS) analyses, no statistically significant associations were identified between the presence of *G. duodenalis*, *Blastocystis* sp. and *E. histolytica*, alone or in combination, and the occurrence of clinical manifestations (Stone-Geisser's Q2 test value <0.0975). Concerning the other predictor, age was not statistically associated with symptomatology in the study population either.

Furthermore, the presence of any of the three protist species investigated, jointly or separately, covaried negatively with symptomatology. The analysis revealed that most of the X's component variance was due to coinfection with enteric protists (43.1%), followed by *Blastocystis* sp. (27.6%) and *E. histolytica* (24.0%) (Table 3).

**Table 3.** Predictor weights of the PLS model explaining their association with clinical symptomatology in hospital-based patients in Tengchong City.

